Methods of detecting nucleic acids in individual cells and of identifying rare cells from large heterogeneous cell populations

ABSTRACT

Methods of detecting multiple nucleic acid targets in single cells through indirect capture of labels to the nucleic acids are provided. Methods of assaying the relative levels of nucleic acid targets through normalization to levels of reference nucleic acids are also provided. Methods of detecting individual cells, particularly rare cells from large heterogeneous cell populations, through detection of nucleic acids are described. Related compositions, systems, and kits are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 16/294,864, filed on Mar. 6, 2019, which is a continuation of U.S.application Ser. No. 11/471,278, filed on Jun. 19, 2006, which is anon-provisional utility patent application claiming priority to andbenefit of the following prior provisional patent application: U.S. Ser.No. 60/691,834, filed on Jun. 20, 2005, entitled “Method of Detectingand Enumerating Rare Cells from Large Heterogeneous Cell Populations” byLuo and Chen, each of which is incorporated herein by reference in itsentirety for all purposes.

FIELD OF THE INVENTION

The invention relates generally to nucleic acid chemistry andbiochemical assays. More particularly, the invention relates to methodsfor in situ detection of nucleic acid analytes in single cells. Theinvention also relates to detection and identification of single cells,particularly rare cells.

BACKGROUND OF THE INVENTION

Ample evidence has demonstrated that cancer cells can dissociate fromthe primary tumor and circulate in the lymph node, bone marrow,peripheral blood or other body fluids. These circulating tumor cells(CTC) have been shown to reflect the biological characteristics of theprimary tumors, including the potential for metastasis development andtumor recurrence. Therefore, the detection of CTC may indicate diseaserecurrence, tumor cell spreading, and a high potential for distantmetastasis. All of these are significant informative clinical factors inidentifying high-risk cancer patients' disease status (e.g. Vogel etal., 2002; Gilbey et al., 2004; Molnar et al., 2003; Vlems et al., 2003;Ma et al., 2003).

Validation of the clinical utility of CTC detection as a prognosticindicator has not been progressing as fast as expected, in large partdue to lack of suitable detection technologies. One key difficulty indetecting CTC in peripheral blood or other body fluids is that CTC arepresent in the circulation in extremely low concentrations, estimated tobe in the range of one tumor cell among 10⁶-10⁷ normal white bloodcells. As a result, any detection technology for this application has toexhibit exceptional sensitivity and specificity in order to limit bothfalse negative and false positive rate to an acceptable level.

One existing approach incorporates immunomagnetic separation technologyin detection of intact CTC (U.S. Pat. Nos. 6,365,362; 6,645,731). Usingthis technology, a blood sample from a cancer patient is incubated withmagnetic beads coated with antibodies directed against an epithelialsurface antigen as for example EpCAM (Cristofanilli et al., 2004). Themagnetically labeled cells are then isolated using a magnetic separator.The immunomagnetically-enriched fraction is further processed fordownstream analysis for CTC identification. Using this technology, itwas shown in a prospective study that the number of CTC after treatmentis an independent predictor of progression-free survival and overallsurvival in patients with metastatic breast cancer (Cristofanilli etal., 2004). Although this technology has reported high sensitivity, itsapplicability is limited by the availability of detection antibodiesthat are highly sensitive and specific to particular types of CTC. Theantibodies can exhibit non-specific binding to other cellular componentswhich can lead to low signal to noise ratio and impair later detection.The antibodies binding to CTC may also bind to antigen present in othertypes of cells at low level, resulting in a high level of falsepositives.

Another approach for determining the presence of CTC has been to testfor the tumor cell specific expression of messenger RNA in blood. Realtime reverse transcription-polymerase chain reaction (QPCR) has beenused to correlate the detection of CTC with patient prognosis. Real-timeRT-PCR has been used for detecting CEA mRNA in peripheral blood ofcolorectal cancer patients (Ito et al., 2002). Disease free survival ofpatients with positive CEA mRNA in post-operative blood wassignificantly shorter than in cases that were negative for CEA mRNA.These results suggest that tumor cells were shed into the bloodstreamand resulted in poor patient outcomes in patients with colorectalcancer. Another report demonstrated the clinical utility of moleculardetection of CTC in high-risk AJCC stage IIBC and IIIAB melanomapatients using multiple mRNA markers by QPCR (Mocellin et al., 2004).The advantage of detecting tumor specific mRNA expression is that anytumor-specific gene can be used to serve as a diagnostic/prognosticmarker. However, the QPCR approach requires the laborious procedure ofmRNA isolation from the blood sample and reverse transcription beforethe PCR reaction. False positives are often observed using thistechnique due to sample contamination by chromosomal DNA or low-levelexpression of the chosen marker gene in normal blood cells (Fava et al.2001). In addition, the limit of detection sensitivity of this techniqueis at most about one tumor cell per 1 ml of blood, and the technologycannot provide an accurate count of CTC numbers.

It is highly desirable to detect and quantitate tumor cell specific mRNAexpression at a single cell level in blood or other body fluids. Atechnology that can detect expression of multiple specific mRNAs inindividual cells in suspension would allow both sensitive and specificdetection and enumeration of CTC in blood or other body fluids. Inaddition, such technology could enable the collection of CTC cells fordownstream cytological and molecular analysis. Currently availabletechniques do not fulfill these needs.

In situ hybridization (ISH) technology is an established method oflocalizing and detecting specific mRNA sequences in morphologicallypreserved tissue sections or cell preparations (Hicks et al., 2004). Themost common specimens used are frozen sections, paraffin embeddedsections or suspension cells that were cytospun onto glass slides andfixed with methanol. Detection is carried out using nucleic acid probesthat are complementary to and hybridize with specific nucleotidesequences within cells and tissues. The sensitivity of the technique issuch that threshold levels of detection are in the range of 10-20 copiesof mRNA per cell.

However, ISH technology faces a number of technical challenges thatlimit its wide use. First of all, cells immobilized on solid surfaceexhibit poor hybridization kinetics. Secondly, assay optimization isgenerally required for a target mRNA in probe selection, labeling, anddetection, for each tissue section in fixation and permeabilization, andin hybridization and washing. In addition, various experiments need tobe performed to control for the specificity of the probe, for tissuemRNA quality, and for the hybridization efficacy of the experimentalprocedure. In addition to technical issues, current ISH technology hasrelatively low performance standards in term of its detectionsensitivity and reproducibility. The false positive rate is still highunless the relevant cells are re-examined manually using theirmorphology, which is time and labor-intensive. Current ISH technologyalso does not have the capability to quantitatively determine the mRNAexpression level or to simultaneously measure the expression of multipletarget mRNA within cells, which may provide clinical valuableinformation such as increased detection sensitivity and specificity, andthe identification of primary tumor type, source and stage.

There are four main types of probes that are typically used inperforming in situ hybridization within cells: oligonucleotide probes(usually 20-40 bases in length), single-stranded DNA probes (200-500bases in length), double stranded DNA probes, or RNA probes (200-5000bases in length). RNA probes are currently the most widely used probesfor in situ hybridization as they have the advantage that RNA-RNAhybrids are very thermostable and are resistant to digestion by RNases.RNA probe is a direct labeling method that suffers a number ofdifficulties. First, separate labeled probes have to be prepared fordetecting each mRNA of interest. Second, it is technically difficult todetect the expression of multiple mRNA of interest in situ at the sametime. As a result, only sequential detection of multiple mRNAs usingdifferent labeling methods has recently been reported (Schrock et al,1996; Kosman et al, 2004). Furthermore, with direct labeling methods,there is no good way to control for potential cross-hybridization withnon-specific sequences in cells. Branched DNA (bDNA) in situhybridization is an indirect labeling method for detecting mRNA insingle cells (Player et al, 2001). This method uses a series ofoligonucleotide probes that have one portion hybridizing to the specificmRNA of interest and another portion hybridizing to the bDNA for signalamplification and detection. bDNA ISH has the advantage of usingunlabeled oligonucleotide probes for detecting every mRNA of interestand the signal amplification and detection are generic components in theassay. However, the gene specific probes in the bDNA ISH need to betheoretically screened against possible non-specific hybridizationinteractions with other mRNA sequences in the cells. The nonspecifichybridization of the oligonucleotide probes in bDNA ISH can become aserious problem when multiple of those probes have to be used for thedetection of low abundance mRNAs. Similarly, although use of bDNA ISH todetect or quantitate multiple mRNAs is desirable, such nonspecifichybridization of the oligonucleotide probes is a potential problem.

The present invention overcomes the above noted difficulties andprovides methods for detecting nucleic acids in and for identifyingindividual cells. A complete understanding of the invention will beobtained upon review of the following.

SUMMARY OF THE INVENTION

Methods of detecting nucleic acid targets in single cells, includingmethods of detecting multiple targets in a single cell, are provided.Methods of detecting individual cells, particularly rare cells fromlarge heterogeneous cell populations, through detection of nucleic acidsare described. Related compositions, systems, and kits are alsodescribed.

A first general class of embodiments includes methods of detecting twoor more nucleic acid targets in an individual cell. In the methods, asample comprising the cell is provided. The cell comprises, or issuspected of comprising, a first nucleic acid target and a secondnucleic acid target. A first label probe comprising a first label and asecond label probe comprising a second label, wherein a first signalfrom the first label is distinguishable from a second signal from thesecond label, are provided. At least a first capture probe and at leasta second capture probe are also provided.

The first capture probe is hybridized, in the cell, to the first nucleicacid target (when the first nucleic acid target is present in the cell),and the second capture probe is hybridized, in the cell, to the secondnucleic acid target (when the second nucleic acid target is present inthe cell). The first label probe is captured to the first capture probeand the second label probe is captured to the second capture probe,thereby capturing the first label probe to the first nucleic acid targetand the second label probe to the second nucleic acid target. The firstsignal from the first label and the second signal from the second labelare then detected. Since the first and second labels are associated withtheir respective nucleic acid targets through the capture probes,presence of the label(s) in the cell indicates the presence of thecorresponding nucleic acid target(s) in the cell. The methods areoptionally quantitative. Thus, an intensity of the first signal and anintensity of the second signal can be measured, and the intensity of thefirst signal can be correlated with a quantity of the first nucleic acidtarget in the cell while the intensity of the second signal iscorrelated with a quantity of the second nucleic acid target in thecell.

In one aspect, the label probes bind directly to the capture probes. Forexample, in one class of embodiments, a single first capture probe and asingle second capture probe are provided, the first label probe ishybridized to the first capture probe, and the second label probe ishybridized to the second capture probe. In a related class ofembodiments, two or more first capture probes and two or more secondcapture probes are provided, as are a plurality of the first labelprobes (e.g., two or more identical first label probes) and a pluralityof the second label probes (e.g., two or more identical second labelprobes). The two or more first capture probes are hybridized to thefirst nucleic acid target, and the two or more second capture probes arehybridized to the second nucleic acid target. A single first label probeis hybridized to each of the first capture probes, and a single secondlabel probe is hybridized to each of the second capture probes.

In another aspect, the label probes are captured to the capture probesindirectly, for example, through binding of preamplifiers and/oramplifiers. In one class of embodiments in which amplifiers areemployed, a single first capture probe, a single second capture probe, aplurality of the first label probes, and a plurality of the second labelprobes are provided. A first amplifier is hybridized to the firstcapture probe and to the plurality of first label probes, and a secondamplifier is hybridized to the second capture probe and to the pluralityof second label probes. In another class of embodiments, two or morefirst capture probes, two or more second capture probes, a plurality ofthe first label probes, and a plurality of the second label probes areprovided. The two or more first capture probes are hybridized to thefirst nucleic acid target, and the two or more second capture probes arehybridized to the second nucleic acid target. A first amplifier ishybridized to each of the first capture probes, and the plurality offirst label probes is hybridized to the first amplifiers. A secondamplifier is hybridized to each of the second capture probes, and theplurality of second label probes is hybridized to the second amplifiers.

In one class of embodiments in which preamplifiers are employed, asingle first capture probe, a single second capture probe, a pluralityof the first label probes, and a plurality of the second label probesare provided. A first preamplifier is hybridized to the first captureprobe, a plurality of first amplifiers is hybridized to the firstpreamplifier, and the plurality of first label probes is hybridized tothe first amplifiers. A second preamplifier is hybridized to the secondcapture probe, a plurality of second amplifiers is hybridized to thesecond preamplifier, and the plurality of second label probes ishybridized to the second amplifiers. In another class of embodiments,two or more first capture probes, two or more second capture probes, aplurality of the first label probes, and a plurality of the second labelprobes are provided. The two or more first capture probes are hybridizedto the first nucleic acid target, and the two or more second captureprobes are hybridized to the second nucleic acid target. A firstpreamplifier is hybridized to each of the first capture probes, aplurality of first amplifiers is hybridized to each of the firstpreamplifiers, and the plurality of first label probes is hybridized tothe first amplifiers. A second preamplifier is hybridized to each of thesecond capture probes, a plurality of second amplifiers is hybridized toeach of the second preamplifiers, and the plurality of second labelprobes is hybridized to the second amplifiers.

In embodiments in which two or more first capture probes and/or two ormore second capture probes are employed, the capture probes preferablyhybridize to nonoverlapping polynucleotide sequences in their respectivenucleic acid target.

In one class of embodiments, a plurality of the first label probes and aplurality of the second label probes are provided. A first amplifiedpolynucleotide is produced by rolling circle amplification of a firstcircular polynucleotide hybridized to the first capture probe. The firstcircular polynucleotide comprises at least one copy of a polynucleotidesequence identical to a polynucleotide sequence in the first labelprobe, and the first amplified polynucleotide thus comprises a pluralityof copies of a polynucleotide sequence complementary to thepolynucleotide sequence in the first label probe. The plurality of firstlabel probes is then hybridized to the first amplified polynucleotide.Similarly, a second amplified polynucleotide is produced by rollingcircle amplification of a second circular polynucleotide hybridized tothe second capture probe. The second circular polynucleotide comprisesat least one copy of a polynucleotide sequence identical to apolynucleotide sequence in the second label probe, and the secondamplified polynucleotide thus comprises a plurality of copies of apolynucleotide sequence complementary to the polynucleotide sequence inthe second label probe. The plurality of second label probes is thenhybridized to the second amplified polynucleotide. The amplifiedpolynucleotides remain associated with the capture probe(s), and thelabel probes are thus captured to the nucleic acid targets.

The methods are useful for multiplex detection of nucleic acids,including simultaneous detection of more than two nucleic acid targets.Thus, the cell optionally comprises or is suspected of comprising athird nucleic acid target, and the methods optionally include: providinga third label probe comprising a third label, wherein a third signalfrom the third label is distinguishable from the first and secondsignals, providing at least a third capture probe, hybridizing in thecell the third capture probe to the third nucleic acid target (whenpresent in the cell), capturing the third label probe to the thirdcapture probe, and detecting the third signal from the third label.Fourth, fifth, sixth, etc. nucleic acid targets are similarlysimultaneously detected in the cell if desired. Each hybridization orcapture step is preferably accomplished for all of the nucleic acidtargets at the same time.

A nucleic acid target can be essentially any nucleic acid that isdesirably detected in the cell. For example, a nucleic acid target canbe a DNA, a chromosomal DNA, an RNA, an mRNA, a microRNA, a ribosomalRNA, or the like. The nucleic acid target can be a nucleic acidendogenous to the cell. As another example, the target can be a nucleicacid introduced to or expressed in the cell by infection of the cellwith a pathogen, for example, a viral or bacterial genomic RNA or DNA, aplasmid, a viral or bacterial mRNA, or the like.

The first and second (and/or optional third, fourth, etc.) nucleic acidtargets can be part of a single nucleic acid molecule, or they can beseparate molecules. In one class of embodiments, the first nucleic acidtarget is a first mRNA and the second nucleic acid target is a secondmRNA. In another class of embodiments, the first nucleic acid targetcomprises a first region of an mRNA and the second nucleic acid targetcomprises a second region of the same mRNA. In another class ofembodiments, the first nucleic acid target comprises a first chromosomalDNA polynucleotide sequence and the second nucleic acid target comprisesa second chromosomal DNA polynucleotide sequence. The first and secondchromosomal DNA polynucleotide sequences are optionally located on thesame chromosome, e.g., within the same gene, or on differentchromosomes.

In one aspect, the signal(s) from nucleic acid target(s) are normalized.In one class of embodiments, the second nucleic acid target comprises areference nucleic acid, and the method includes normalizing the firstsignal to the second signal. The label (first, second, third, etc.) canbe essentially any convenient label that directly or indirectly providesa detectable signal. In one aspect, the first label is a firstfluorescent label and the second label is a second fluorescent label.

The methods can be used to detect the presence of the nucleic acidtargets in cells from essentially any type of sample. For example, thesample can be derived from a bodily fluid such as blood. The methods fordetecting nucleic acid targets in cells can be used to identify thecells. For example, a cell can be identified as being of a desired typebased on which nucleic acids, and in what levels, it contains. Thus, inone class of embodiments, the methods include identifying the cell as adesired target cell based on detection of the first and second signals(and optional third, fourth, etc. signals) from within the cell. As justa few examples, the cell can be a circulating tumor cell, a virallyinfected cell, a fetal cell in maternal blood, a bacterial cell or othermicroorganism in a biological sample, or an endothelial cell, precursorendothelial cell, or myocardial cell in blood.

The cell is typically fixed and permeabilized before hybridization ofthe capture probes, to retain the nucleic acid targets in the cell andto permit the capture probes, label probes, etc. to enter the cell. Thecell is optionally washed to remove materials not captured to one of thenucleic acid targets. The cell can be washed after any of various steps,for example, after hybridization of the capture probes to the nucleicacid targets to remove unbound capture probes, after hybridization ofthe preamplifiers, amplifiers, and/or label probes to the captureprobes, and/or the like. It will be evident that double-stranded nucleicacid target(s) are preferably denatured, e.g., by heat, prior tohybridization of the corresponding capture probe(s) to the target(s).

Preferably, the cell is in suspension for all or most of the steps ofthe method. Thus, in one class of embodiments, the cell is in suspensionin the sample comprising the cell, and/or the cell is in suspensionduring the hybridizing, capturing, and/or detecting steps. In otherembodiments, the cell is in suspension in the sample comprising thecell, and the cell is fixed on a substrate during the hybridizing,capturing, and/or detecting steps. For example, the cell can be insuspension during the hybridization, capturing, and optional washingsteps and immobilized on a substrate during the detection step. Inembodiments in which the cell is in suspension, the first and second(and optional third, etc.) signals can be conveniently detected by flowcytometry. Signals from the labels are typically detected in a singleoperation.

One general class of embodiments provides methods of assaying a relativelevel of one or more target nucleic acids in an individual cell. In themethods, a sample comprising the cell is provided. The cell comprises oris suspected of comprising a first, target nucleic acid, and itcomprises a second, reference nucleic acid. A first label probecomprising a first label and a second label probe comprising a secondlabel, wherein a first signal from the first label is distinguishablefrom a second signal from the second label, are also provided. In thecell, the first label probe is captured to the first, target nucleicacid (when present in the cell) and the second label probe is capturedto the second, reference nucleic acid. The first signal from the firstlabel and the second signal from the second label are then detected inthe individual cell, and the intensity of each signal is measured. Theintensity of the first signal is normalized to the intensity of thesecond (reference) signal. The level of the first, target nucleic acidrelative to the level of the second, reference nucleic acid in the cellis thereby assayed, since the first and second labels are associatedwith their respective nucleic acids. The methods are optionallyquantitative, permitting measurement of the amount of the first, targetnucleic acid relative to the amount of the second, reference nucleicacid in the cell. Thus, the intensity of the first signal normalized tothat of the second signal can be correlated with a quantity of thefirst, target nucleic acid present in the cell.

The label probes can bind directly to the nucleic acids. For example,the first label probe can hybridize to the first, target nucleic acidand/or the second label probe can hybridize to the second, referencenucleic acid. Alternatively, the label probes can be bound indirectly tothe nucleic acids, e.g., via capture probes. In one class ofembodiments, at least a first capture probe and at least a secondcapture probe are provided. In the cell, the first capture probe ishybridized to the first, target nucleic acid and the second captureprobe is hybridized to the second, reference nucleic acid. The firstlabel probe is captured to the first capture probe and the second labelprobe is captured to the second capture probe, thereby capturing thefirst label probe to the first, target nucleic acid and the second labelprobe to the second, reference nucleic acid. The features described forthe methods above apply to these embodiments as well, with respect toconfiguration and number of the label and capture probes, optional useof preamplifiers and/or amplifiers, rolling circle amplification ofcircular polynucleotides, and the like.

The methods can be used for multiplex detection of nucleic acids,including simultaneous detection of two or more target nucleic acids.Thus, the cell optionally comprises or is suspected of comprising athird, target nucleic acid, and the methods optionally include:providing a third label probe comprising a third label, wherein a thirdsignal from the third label is distinguishable from the first and secondsignals; capturing, in the cell, the third label probe to the third,target nucleic acid (when present in the cell); detecting the thirdsignal from the third label, which detecting comprises measuring anintensity of the third signal; and normalizing the intensity of thethird signal to the intensity of the second signal. Fourth, fifth,sixth, etc. nucleic acids are similarly simultaneously detected in thecell if desired.

The methods for assaying relative levels of target nucleic acids incells can be used to identify the cells. For example, a cell can beidentified as being of a desired type based on which nucleic acids, andin what levels, it contains. Thus, in one class of embodiments, themethods include identifying the cell as a desired target cell based onthe normalized first signal (and optional normalized third, fourth, etc.signals).

Essentially all of the features noted for the methods above apply tothese embodiments as well, as relevant; for example, with respect totype of target and reference nucleic acids, cell type, source of sample,fixation and permeabilization of the cell, washing the cell,denaturation of double-stranded target and reference nucleic acids, typeof labels, use of optional blocking probes, detection of signals,detection (and intensity measurement) by flow cytometry or microscopy,presence of the cell in suspension or immobilized on a substrate, and/orthe like.

Another general class of embodiments provides methods of performingcomparative gene expression analysis in single cells. In the methods, afirst mixed cell population comprising one or more cells of a specifiedtype is provided. An expression level of one or more target nucleicacids relative to a reference nucleic acid is measured in the cells ofthe specified type of the first population, to provide a firstexpression profile. A second mixed cell population comprising one ormore cells of the specified type is also provided, and an expressionlevel of the one or more target nucleic acids relative to the referencenucleic acid is measured in the cells of the specified type of thesecond population, to provide a second expression profile. The first andsecond expression profiles are then compared.

Essentially all of the features noted for the methods above apply tothese embodiments as well, as relevant; for example, with respect totype of target and reference nucleic acids, cell type, source of sample,fixation and permeabilization of the cell, washing the cell,denaturation of double-stranded target and reference nucleic acids, typeof labels, use and configuration of label probes, capture probes,preamplifiers and/or amplifiers, use of optional blocking probes,detection of signals, detection (and intensity measurement) by flowcytometry or microscopy, presence of the cell in suspension orimmobilized on a substrate, and/or the like.

In one aspect, the invention provides methods that facilitateassociation of a high density of labels to target nucleic acids incells. One general class of embodiments provides methods of detectingtwo or more nucleic acid targets in an individual cell. In the methods,a sample comprising the cell is provided. The cell comprises or issuspected of comprising a first nucleic acid target and a second nucleicacid target. In the cell, a first label is captured to the first nucleicacid target (when present in the cell) and a second label is captured tothe second nucleic acid target (when present in the cell). A firstsignal from the first label is distinguishable from a second signal fromthe second label. As noted, the labels are captured at high density.Thus, an average of at least one copy of the first label per nucleotideof the first nucleic acid target is captured to the first nucleic acidtarget over a region that spans at least 20 contiguous nucleotides ofthe first nucleic acid target, and an average of at least one copy ofthe second label per nucleotide of the second nucleic acid target iscaptured to the second nucleic acid target over a region that spans atleast 20 contiguous nucleotides of the second nucleic acid target. Thefirst signal from the first label and the second signal from the secondlabel are detected.

In one class of embodiments, an average of at least four, eight, ortwelve copies of the first label per nucleotide of the first nucleicacid target are captured to the first nucleic acid target over a regionthat spans at least 20 contiguous nucleotides of the first nucleic acidtarget, and an average of at least four, eight, or twelve copies of thesecond label per nucleotide of the second nucleic acid target arecaptured to the second nucleic acid target over a region that spans atleast 20 contiguous nucleotides of the second nucleic acid target. Inone embodiment, an average of at least sixteen copies of the first labelper nucleotide of the first nucleic acid target are captured to thefirst nucleic acid target over a region that spans at least 20contiguous nucleotides of the first nucleic acid target, and an averageof at least sixteen copies of the second label per nucleotide of thesecond nucleic acid target are captured to the second nucleic acidtarget over a region that spans at least 20 contiguous nucleotides ofthe second nucleic acid target.

Essentially all of the features noted for the methods above apply tothese embodiments as well, as relevant, for example, with respect totype of labels, detection of signals, type, treatment, and suspension ofthe cell, and/or the like. A like density of third, fourth, fifth,sixth, etc. labels is optionally captured to third, fourth, fifth,sixth, etc. nucleic acid targets.

Another general class of embodiments provides methods of detecting anindividual cell of a specified type. In the methods, a sample comprisinga mixture of cell types including at least one cell of the specifiedtype is provided. A first label probe comprising a first label and asecond label probe comprising a second label, wherein a first signalfrom the first label is distinguishable from a second signal from thesecond label, are provided. In the cell, the first label probe iscaptured to a first nucleic acid target (when the first nucleic acidtarget is present in the cell) and the second label probe is captured toa second nucleic acid target (when the second nucleic acid target ispresent in the cell). The first signal from the first label and thesecond signal from the second label are detected and correlated with thepresence, absence, or amount of the corresponding, first and secondnucleic acid targets in the cell. The cell is identified as being of thespecified type based on detection of the presence, absence, or amount ofboth the first and second nucleic acid targets within the cell, wherethe specified type of cell is distinguishable from the other celltype(s) in the mixture on the basis of either the presence, absence, oramount of the first nucleic acid target or the presence, absence, oramount of the second nucleic acid target in the cell (that is, thenucleic acid targets are redundant markers for the specified cell type).An intensity of the first signal and an intensity of the second signalare optionally measured and correlated with a quantity of thecorresponding nucleic acid present in the cell. In one class ofembodiments, the cell comprises a first nucleic acid target and a secondnucleic acid target, and the cell is identified as being of thespecified type based on detection of the presence or amount of both thefirst and second nucleic acid targets within the cell, where thespecified type of cell is distinguishable from the other cell type(s) inthe mixture on the basis of either the presence or amount of the firstnucleic acid target or the presence or amount of the second nucleic acidtarget in the cell.

The label probes can bind directly to the nucleic acid targets. Forexample, the first label probe can hybridize to the first nucleic acidtarget and/or the second label probe can hybridize to the second nucleicacid target. The label probes are optionally captured to the nucleicacid targets via capture probes. In one class of embodiments, at least afirst capture probe and at least a second capture probe are provided. Inthe cell, the first capture probe is hybridized to the first nucleicacid target and the second capture probe is hybridized to the secondnucleic acid target. The first label probe is captured to the firstcapture probe and the second label probe is captured to the secondcapture probe, thereby capturing the first label probe to the firstnucleic acid target and the second label probe to the second nucleicacid target. The features described for the methods above apply to theseembodiments as well, with respect to configuration and number of thelabel and capture probes, optional use of preamplifiers and/oramplifiers, rolling circle amplification of circular polynucleotides,and the like.

Third, fourth, fifth, etc. nucleic acid targets are optionally detectedin the cell. For example, the method optionally includes: providing athird label probe comprising a third label, wherein a third signal fromthe third label is distinguishable from the first and second signals,capturing in the cell the third label probe to a third nucleic acidtarget (when the third target is present in the cell), and detecting thethird signal from the third label. The third, fourth, fifth, etc. labelprobes are optionally hybridized directly to their corresponding nucleicacid, or they can be captured indirectly via capture probes as describedfor the first and second label probes.

The first and/or second signal can be normalized to the third signal.Thus, in some embodiments, the cell comprises the third nucleic acidtarget, and the methods include identifying the cell as being of thespecified type based on the normalized first and/or second signal, e.g.,in embodiments in which the target cell type is distinguishable from theother cell type(s) in the mixture based on the copy number of the firstand/or second nucleic acid targets, rather than purely on their presencein the target cell type and not in the other cell type(s).

As another example, the third nucleic acid target can serve as a thirdredundant marker for the target cell type, e.g., to improve specificityof the assay for the desired cell type. Thus, in one class ofembodiments, the methods include correlating the third signal detectedfrom the cell with the presence, absence, or amount of the third nucleicacid target in the cell, and identifying the cell as being of thespecified type based on detection of the presence, absence, or amount ofthe first, second, and third nucleic acid targets within the cell,wherein the specified type of cell is distinguishable from the othercell type(s) in the mixture on the basis of either presence, absence, oramount of the first nucleic acid target, presence, absence, or amount ofthe second nucleic acid target, or presence, absence, or amount of thethird nucleic acid target in the cell.

The methods can be applied to detection and identification of even rarecell types. For example, the ratio of cells of the specified type tocells of all other type(s) in the mixture is optionally less than1:1×10⁴, less than 1:1×10⁵, less than 1:1×10⁶, less than 1:1×10⁷, lessthan 1:1×10⁸, or even less than 1:1×10⁹.

Essentially all of the features noted for the methods above apply tothese embodiments as well, as relevant; for example, with respect totype of nucleic acid targets, cell type, source of sample, fixation andpermeabilization of the cell, washing the cell, denaturation ofdouble-stranded nucleic acids, type of labels, use of optional blockingprobes, detection of signals, detection (and intensity measurement) ofsignals from the individual cell by flow cytometry or microscopy,presence of the cell in suspension or immobilized on a substrate, and/orthe like.

The invention also provides compositions useful in practicing orproduced by the methods. One exemplary class of embodiments provides acomposition that includes a fixed and permeabilized cell, which cellcomprises or is suspected of comprising a first nucleic acid target anda second nucleic acid target, at least a first capture probe capable ofhybridizing to the first nucleic acid target, at least a second captureprobe capable of hybridizing to the second nucleic acid target, a firstlabel probe comprising a first label, and a second label probecomprising a second label. A first signal from the first label isdistinguishable from a second signal from the second label. The celloptionally comprises the first and second capture probes and labelprobes. The first and second capture probes are optionally hybridized totheir respective nucleic acid targets in the cell.

The features described for the methods above for indirect capture of thelabel probes to the nucleic acid targets apply to these embodiments aswell, for example, with respect to configuration and number of the labeland capture probes, optional use of preamplifiers and/or amplifiers, andthe like.

In one class of embodiments, the composition comprises a plurality ofthe first label probes, a plurality of the second label probes, a firstamplified polynucleotide produced by rolling circle amplification of afirst circular polynucleotide hybridized to the first capture probe, anda second amplified polynucleotide produced by rolling circleamplification of a second circular polynucleotide hybridized to thesecond capture probe. The first circular polynucleotide comprises atleast one copy of a polynucleotide sequence identical to apolynucleotide sequence in the first label probe, and the firstamplified polynucleotide comprises a plurality of copies of apolynucleotide sequence complementary to the polynucleotide sequence inthe first label probe. The second circular polynucleotide comprises atleast one copy of a polynucleotide sequence identical to apolynucleotide sequence in the second label probe, and the secondamplified polynucleotide comprises a plurality of copies of apolynucleotide sequence complementary to the polynucleotide sequence inthe second label probe. The composition can also include reagentsnecessary for producing the amplified polynucleotides, for example, anexogenously supplied nucleic acid polymerase, an exogenously suppliednucleic acid ligase, and/or exogenously supplied nucleosidetriphosphates (e.g., dNTPs).

The cell optionally includes additional nucleic acid targets, and thecomposition (and cell) can include reagents for detecting these targets.For example, the cell can comprise or be suspected of comprising a thirdnucleic acid target, and the composition can include at least a thirdcapture probe capable of hybridizing to the third nucleic acid targetand a third label probe comprising a third label. A third signal fromthe third label is distinguishable from the first and second signals.The cell optionally includes fourth, fifth, sixth, etc. nucleic acidtargets, and the composition optionally includes fourth, fifth, sixth,etc. label probes and capture probes.

The cell can be present in a mixture of cells, for example, a complexheterogeneous mixture. In one class of embodiments, the cell is of aspecified type, and the composition comprises one or more other types ofcells. These other cells can be present in excess, even large excess, ofthe cell. For example, the ratio of cells of the specified type to cellsof all other type(s) in the composition is optionally less than 1:1×10⁴,less than 1:1×10⁵, less than 1:1×10⁶, less than 1:1×10⁷, less than1:1×10⁸, or even less than 1:1×10⁹.

Essentially all of the features noted for the methods above apply tothese embodiments as well, as relevant; for example, with respect totype of nucleic acid target, type and source of cell, location ofvarious targets on a single molecule or on different molecules, type oflabels, inclusion of optional blocking probes, and/or the like. The cellis optionally in suspension in the composition.

One general class of embodiments provides a composition comprising acell, which cell includes a first nucleic acid target, a second nucleicacid target, a first label whose presence in the cell is indicative ofthe presence of the first nucleic acid target in the cell, and a secondlabel whose presence in the cell is indicative of the presence of thesecond nucleic acid target in the cell, wherein a first signal from thefirst label is distinguishable from a second signal from the secondlabel. An average of at least one copy of the first label is present inthe cell per nucleotide of the first nucleic acid target over a regionthat spans at least 20 contiguous nucleotides of the first nucleic acidtarget, and an average of at least one copy of the second label ispresent in the cell per nucleotide of the second nucleic acid targetover a region that spans at least 20 contiguous nucleotides of thesecond nucleic acid target.

In one class of embodiments, the copies of the first label arephysically associated with the first nucleic acid target, and the copiesof the second label are physically associated with the second nucleicacid target. For example, the first label can be part of a first labelprobe and the second label part of a second label probe, where the labelprobes are captured to the target nucleic acids.

Essentially all of the features noted for the embodiments above apply tothese embodiments as well, as relevant, for example, with respect totype and number of labels, suspension of the cell, and/or the like. Alike density of labels is optionally present for third, fourth, fifth,sixth, etc. nucleic acid targets.

Another aspect of the invention provides kits useful for practicing themethods. One general class of embodiments provides a kit for detecting afirst nucleic acid target and a second nucleic acid target in anindividual cell. The kit includes at least one reagent for fixing and/orpermeabilizing the cell, at least a first capture probe capable ofhybridizing to the first nucleic acid target, at least a second captureprobe capable of hybridizing to the second nucleic acid target, a firstlabel probe comprising a first label, and a second label probecomprising a second label, wherein a first signal from the first labelis distinguishable from a second signal from the second label, packagedin one or more containers.

Essentially all of the features noted for the embodiments above apply tothese embodiments as well, as relevant; for example, with respect tonumber of nucleic acid targets, configuration and number of the labeland capture probes, inclusion of preamplifiers and/or amplifiers,inclusion of blocking probes, inclusion of amplification reagents, typeof nucleic acid target, location of various targets on a single moleculeor on different molecules, type of labels, inclusion of optionalblocking probes, and/or the like.

Another general class of embodiments provides a kit for detecting anindividual cell of a specified type from a mixture of cell types bydetecting a first nucleic acid target and a second nucleic acid target.The kit includes at least one reagent for fixing and/or permeabilizingthe cell, a first label probe comprising a first label, and a secondlabel probe comprising a second label, wherein a first signal from thefirst label is distinguishable from a second signal from the secondlabel, packaged in one or more containers. The specified type of cell isdistinguishable from the other cell type(s) in the mixture by presence,absence, or amount of the first nucleic acid target in the cell or bypresence, absence, or amount of the second nucleic acid target in thecell.

Essentially all of the features noted for the embodiments above apply tothese embodiments as well, as relevant; for example, with respect tonumber of nucleic acid targets, inclusion of capture probes,configuration and number of the label and/or capture probes, inclusionof preamplifiers and/or amplifiers, inclusion of blocking probes,inclusion of amplification reagents, type of nucleic acid target,location of various targets on a single molecule or on differentmolecules, type of labels, inclusion of optional blocking probes, and/orthe like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates QMAGEX technology workflow for anexemplary embodiment.

FIG. 2 schematically illustrates a direct labeling approach in whichlabel probes are hybridized to the target nucleic acid.

FIG. 3 schematically illustrates an indirect labeling approach in whichlabel probes are hybridized to capture probes hybridized to the targetnucleic acid.

FIG. 4 schematically illustrates an indirect labeling capture probedesign approach that utilizes a pair of independent capture probes toenhance the specificity of the label probe capture to the target nucleicacid.

FIG. 5 schematically illustrates an indirect labeling capture probedesign approach that utilizes three or more independent capture probesto enhance the specificity of the label probe capture to the targetnucleic acid.

FIGS. 6A-6B schematically illustrate probe design approaches to detectmultiple target molecules in parallel using either direct labeling (FIG.6A) or indirect labeling with two independent capture probes (FIG. 6B).

FIGS. 7A-7C schematically illustrate probe design approaches to reducingfalse positive rates in rare cell identification by attaching multipletypes of signal-generating particles (labels) to the same targetmolecule. FIG. 7A shows multiple types of signal-generating panicles(labels) on one target. FIG. 7B shows multiple types ofsignal-generating panicles (labels) on more than one target, where therelative signal strengths of the particle set are maintained across alltargets. FIG. 7C shows a set of signal-generating particles (labels) ona target molecule, where different targets have distinctively differentsets.

FIGS. 8A-8D schematically illustrate different structures of exemplaryamplifiers.

FIGS. 9A-9C schematically illustrate utilizing rolling circleamplification to amplify signal. As shown in FIG. 9A, a circularnucleotide molecule is attached to capture probe(s). As shown in FIG.9B, a long chain molecule with many repeated sequences appears as aresult of rolling circle amplification. As shown in FIG. 9C, many signalprobes can be hybridized to the repeated sequences to achieve signalamplification.

FIG. 10 schematically illustrates one embodiment of the assay instrumentconfiguration.

Schematic figures are not necessarily to scale.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. The following definitionssupplement those in the art and are directed to the current applicationand are not to be imputed to any related or unrelated case, e.g., to anycommonly owned patent or application. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice for testing of the present invention, the preferred materialsand methods are described herein. Accordingly, the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a molecule”includes a plurality of such molecules, and the like.

The term “about” as used herein indicates the value of a given quantityvaries by +/−10% of the value, or optionally +/−5% of the value, or insome embodiments, by +/−1% of the value so described.

The term “polynucleotide” (and the equivalent term “nucleic acid”)encompasses any physical string of monomer units that can becorresponded to a string of nucleotides, including a polymer ofnucleotides (e.g., a typical DNA or RNA polymer), peptide nucleic acids(PNAs), modified oligonucleotides (e.g., oligonucleotides comprisingnucleotides that are not typical to biological RNA or DNA, such as2′-O-methylated oligonucleotides), and the like. The nucleotides of thepolynucleotide can be deoxyribonucleotides, ribonucleotides ornucleotide analogs, can be natural or non-natural, and can beunsubstituted, unmodified, substituted or modified. The nucleotides canbe linked by phosphodiester bonds, or by phosphorothioate linkages,methylphosphonate linkages, boranophosphate linkages, or the like. Thepolynucleotide can additionally comprise non-nucleotide elements such aslabels, quenchers, blocking groups, or the like. The polynucleotide canbe, e.g., single-stranded or double-stranded.

A “nucleic acid target” or “target nucleic acid” refers to a nucleicacid, or optionally a region thereof, that is to be detected.

A “polynucleotide sequence” or “nucleotide sequence” is a polymer ofnucleotides (an oligonucleotide, a DNA, a nucleic acid, etc.) or acharacter string representing a nucleotide polymer, depending oncontext. From any specified polynucleotide sequence, either the givennucleic acid or the complementary polynucleotide sequence (e.g., thecomplementary nucleic acid) can be determined.

The term “gene” is used broadly to refer to any nucleic acid associatedwith a biological function. Genes typically include coding sequencesand/or the regulatory sequences required for expression of such codingsequences. The term gene can apply to a specific genomic sequence, aswell as to a cDNA or an mRNA encoded by that genomic sequence. Genesalso include non-expressed nucleic acid segments that, for example, formrecognition sequences for other proteins. Non-expressed regulatorysequences include promoters and enhancers, to which regulatory proteinssuch as transcription factors bind, resulting in transcription ofadjacent or nearby sequences.

Two polynucleotides “hybridize” when they associate to form a stableduplex, e.g., under relevant assay conditions. Nucleic acids hybridizedue to a variety of well characterized physico-chemical forces, such ashydrogen bonding, solvent exclusion, base stacking and the like. Anextensive guide to the hybridization of nucleic acids is found inTijssen (1993) Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, part I chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays” (Elsevier, New York), as well as in Ausubel, infra.

A first polynucleotide “capable of hybridizing” to a secondpolynucleotide contains a first polynucleotide sequence that iscomplementary to a second polynucleotide sequence in the secondpolynucleotide. The first and second polynucleotides are able to form astable duplex, e.g., under relevant assay conditions.

The “T_(m)” (melting temperature) of a nucleic acid duplex underspecified conditions (e.g., relevant assay conditions) is thetemperature at which half of the base pairs in a population of theduplex are disassociated and half are associated. The T_(m) for aparticular duplex can be calculated and/or measured, e.g., by obtaininga thermal denaturation curve for the duplex (where the T_(m) is thetemperature corresponding to the midpoint in the observed transitionfrom double-stranded to single-stranded form).

The term “complementary” refers to a polynucleotide that forms a stableduplex with its “complement,” e.g., under relevant assay conditions.Typically, two polynucleotide sequences that are complementary to eachother have mismatches at less than about 20% of the bases, at less thanabout 10% of the bases, preferably at less than about 5% of the bases,and more preferably have no mismatches.

A “label” is a moiety that facilitates detection of a molecule. Commonlabels in the context of the present invention include fluorescent,luminescent, light-scattering, and/or colorimetric labels. Suitablelabels include enzymes and fluorescent moieties, as well asradionuclides, substrates, cofactors, inhibitors, chemiluminescentmoieties, magnetic panicles, and the like. Patents teaching the use ofsuch labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241. Many labels arecommercially available and can be used in the context of the invention.

The term “label probe” refers to an entity that binds to a targetmolecule, directly or indirectly, and enables the target to be detected,e.g., by a readout instrument. A label probe (or “LP”) is typically asingle-stranded polynucleotide that comprises at least one label whichdirectly or indirectly provides a detectable signal. The label can becovalently attached to the polynucleotide, or the polynucleotide can beconfigured to bind to the label (e.g., a biotinylated polynucleotide canbind a streptavidin-associated label). The label probe can, for example,hybridize directly to a target nucleic acid, or it can hybridize to anucleic acid that is in turn hybridized to the target nucleic acid or toone or more other nucleic acids that are hybridized to the nucleic acid.Thus, the label probe can comprise a polynucleotide sequence that iscomplementary to a polynucleotide sequence of the target nucleic acid,or it can comprise at least one polynucleotide sequence that iscomplementary to a polynucleotide sequence in a capture probe,amplifier, or the like.

A “capture probe” is a polynucleotide that is capable of hybridizing toa target nucleic acid and capturing a label probe to that target nucleicacid. The capture probe can hybridize directly to the label probe, or itcan hybridize to one or more nucleic acids that in turn hybridize to thelabel probe; for example, the capture probe can hybridize to anamplifier or a preamplifier. The capture probe thus includes a firstpolynucleotide sequence that is complementary to a polynucleotidesequence of the target nucleic acid and a second polynucleotide sequencethat is complementary to a polynucleotide sequence of the label probe,amplifier, preamplifier, or the like. The capture probe is preferablysingle-stranded.

An “amplifier” is a molecule, typically a polynucleotide, that iscapable of hybridizing to multiple label probes. Typically, theamplifier hybridizes to multiple identical label probes. The amplifieralso hybridizes to at least one capture probe or nucleic acid bound to acapture probe. For example, the amplifier can hybridize to at least onecapture probe and to a plurality of label probes, or to a preamplifierand a plurality of label probes. The amplifier can be, e.g., a linear,forked, comb-like, or branched nucleic acid. As noted for allpolynucleotides, the amplifier can include modified nucleotides and/ornonstandard internucleotide linkages as well as standarddeoxyribonucleotides, ribonucleotides, and/or phosphodiester bonds.Suitable amplifiers are described, for example, in U.S. Pat. Nos.5,635,352, 5,124,246, 5,710,264, and 5,849,481.

A “preamplifier” is a molecule, typically a polynucleotide, that servesas an intermediate between one or more capture probes and amplifiers.Typically, the preamplifier hybridizes simultaneously to one or morecapture probes and to a plurality of amplifiers. Exemplary preamplifiersare described, for example, in U.S. Pat. Nos. 5,635,352 and 5,681,697.

A “pathogen” is a biological agent, typically a microorganism, thatcauses disease or illness to its host.

A “microorganism” is an organism of microscopic or submicroscopic size.Examples include, but are not limited to, bacteria, fungi, yeast,protozoans, microscopic algae (e.g., unicellular algae), viruses (whichare typically included in this category although they are incapable ofgrowth and reproduction outside of host cells), subviral agents,viroids, and mycoplasma.

A variety of additional terms are defined or otherwise characterizedherein.

DETAILED DESCRIPTION

Among other aspects, the present invention provides multiplex assaysthat can be used for simultaneous detection, and optionallyquantitation, of two or more nucleic acid targets in a single cell. Arelated aspect of the invention provides methods for detecting the levelof one or more target nucleic acids relative to that of a referencenucleic acid in an individual cell.

In general, in the assays of the invention, a label probe is captured toeach target nucleic acid. The label probe can be captured to the targetthrough direct binding of the label probe to the target. Preferably,however, the label probe is captured indirectly through binding tocapture probes, amplifiers, and/or preamplifiers that bind to thetarget. Use of the optional amplifiers and preamplifiers facilitatescapture of multiple copies of the label probe to the target, thusamplifying signal from the target without requiring enzymaticamplification of the target itself. Binding of the capture probes isoptionally cooperative, reducing background caused by undesired crosshybridization of capture probes to non-target nucleic acids (a greaterproblem in multiplex assays than singleplex assays since more probesmust be used in multiplex assays, increasing the likelihood of crosshybridization).

One aspect of the invention relates to detection of single cells,including detection of rare cells from a heterogeneous mixture of cells.Individual cells are detected through detection of nucleic acids whosepresence, absence, copy number, or the like are characteristic of thecell.

Compositions, kits, and systems related to the methods are alsoprovided.

Methods of Detecting Nucleic Acids and Cells

Multiplex Detection of Nucleic Acids

As noted, one aspect of the invention provides multiplex nucleic acidassays in single cells. Thus, one general class of embodiments includesmethods of detecting two or more nucleic acid targets in an individualcell. In the methods, a sample comprising the cell is provided. The cellcomprises, or is suspected of comprising, a first nucleic acid targetand a second nucleic acid target. A first label probe comprising a firstlabel and a second label probe comprising a second label, wherein afirst signal from the first label is distinguishable from a secondsignal from the second label, are provided. At least a first captureprobe and at least a second capture probe are also provided.

The first capture probe is hybridized, in the cell, to the first nucleicacid target (when the first nucleic acid target is present in the cell),and the second capture probe is hybridized, in the cell, to the secondnucleic acid target (when the second nucleic acid target is present inthe cell). The first label probe is captured to the first capture probeand the second label probe is captured to the second capture probe,thereby capturing the first label probe to the first nucleic acid targetand the second label probe to the second nucleic acid target. The firstsignal from the first label and the second signal from the second labelare then detected. Since the first and second labels are associated withtheir respective nucleic acid targets through the capture probes,presence of the label(s) in the cell indicates the presence of thecorresponding nucleic acid target(s) in the cell. The methods areoptionally quantitative. Thus, an intensity of the first signal and anintensity of the second signal can be measured, and the intensity of thefirst signal can be correlated with a quantity of the first nucleic acidtarget in the cell while the intensity of the second signal iscorrelated with a quantity of the second nucleic acid target in thecell.

In one aspect, the label probes bind directly to the capture probes. Forexample, in one class of embodiments, a single first capture probe and asingle second capture probe are provided, the first label probe ishybridized to the first capture probe, and the second label probe ishybridized to the second capture probe. In a related class ofembodiments, two or more first capture probes and two or more secondcapture probes are provided, as are a plurality of the first labelprobes (e.g., two or more identical first label probes) and a pluralityof the second label probes (e.g., two or more identical second labelprobes). The two or more first capture probes are hybridized to thefirst nucleic acid target, and the two or more second capture probes arehybridized to the second nucleic acid target. A single first label probeis hybridized to each of the first capture probes, and a single secondlabel probe is hybridized to each of the second capture probes.

In another aspect, the label probes are captured to the capture probesindirectly, for example, through binding of preamplifiers and/oramplifiers. Use of amplifiers and preamplifiers can be advantageous inincreasing signal strength, since they can facilitate binding of largenumbers of label probes to each nucleic acid target.

In one class of embodiments in which amplifiers are employed, a singlefirst capture probe, a single second capture probe, a plurality of thefirst label probes, and a plurality of the second label probes areprovided. A first amplifier is hybridized to the first capture probe andto the plurality of first label probes, and a second amplifier ishybridized to the second capture probe and to the plurality of secondlabel probes. In another class of embodiments, two or more first captureprobes, two or more second capture probes, a plurality of the firstlabel probes, and a plurality of the second label probes are provided.The two or more first capture probes are hybridized to the first nucleicacid target, and the two or more second capture probes are hybridized tothe second nucleic acid target. A first amplifier is hybridized to eachof the first capture probes, and the plurality of first label probes ishybridized to the first amplifiers. A second amplifier is hybridized toeach of the second capture probes, and the plurality of second labelprobes is hybridized to the second amplifiers.

In one class of embodiments in which preamplifiers are employed, asingle first capture probe, a single second capture probe, a pluralityof the first label probes, and a plurality of the second label probesare provided. A first preamplifier is hybridized to the first captureprobe, a plurality of first amplifiers is hybridized to the firstpreamplifier, and the plurality of first label probes is hybridized tothe first amplifiers. A second preamplifier is hybridized to the secondcapture probe, a plurality of second amplifiers is hybridized to thesecond preamplifier, and the plurality of second label probes ishybridized to the second amplifiers. In another class of embodiments,two or more first capture probes, two or more second capture probes, aplurality of the first label probes, and a plurality of the second labelprobes are provided. The two or more first capture probes are hybridizedto the first nucleic acid target, and the two or more second captureprobes are hybridized to the second nucleic acid target. A firstpreamplifier is hybridized to each of the first capture probes, aplurality of first amplifiers is hybridized to each of the firstpreamplifiers, and the plurality of first label probes is hybridized tothe first amplifiers. A second preamplifier is hybridized to each of thesecond capture probes, a plurality of second amplifiers is hybridized toeach of the second preamplifiers, and the plurality of second labelprobes is hybridized to the second amplifiers. Optionally, additionalpreamplifiers can be used as intermediates between a preamplifierhybridized to the capture probe(s) and the amplifiers.

In the above classes of embodiments, one capture probe hybridizes toeach label probe, amplifier, or preamplifier. In alternative classes ofrelated embodiments, two or more capture probes hybridize to the labelprobe, amplifier, or preamplifier. See, e.g., the section below entitled“Implementation, applications, and advantages.”

In embodiments in which two or more first capture probes and/or two ormore second capture probes are employed, the capture probes preferablyhybridize to nonoverlapping polynucleotide sequences in their respectivenucleic acid target. The capture probes can, but need not, cover acontiguous region of the nucleic acid target. Blocking probes,polynucleotides which hybridize to regions of the nucleic acid targetnot occupied by capture probes, are optionally provided and hybridizedto the target. For a given nucleic acid target, the correspondingcapture probes and blocking probes are preferably complementary tophysically distinct, nonoverlapping sequences in the nucleic acidtarget, which nonoverlapping sequences are preferably, but notnecessarily, contiguous. Having the capture probes and optional blockingprobes be contiguous with each other can in some embodiments enhancehybridization strength, remove secondary structure, and ensure moreconsistent and reproducible signal.

In many embodiments, such as those above, enzymatic manipulation is notrequired to capture the label probes to the capture probes. In otherembodiments, however, enzymatic manipulation, particularly amplificationof nucleic acids intermediate between the capture probes and the labelprobes, facilitates detection of the nucleic acid targets. For example,in one class of embodiments, a plurality of the first label probes and aplurality of the second label probes are provided. A first amplifiedpolynucleotide is produced by rolling circle amplification of a firstcircular polynucleotide hybridized to the first capture probe. The firstcircular polynucleotide comprises at least one copy of a polynucleotidesequence identical to a polynucleotide sequence in the first labelprobe, and the first amplified polynucleotide thus comprises a pluralityof copies of a polynucleotide sequence complementary to thepolynucleotide sequence in the first label probe. The plurality of firstlabel probes is then hybridized to the first amplified polynucleotide.Similarly, a second amplified polynucleotide is produced by rollingcircle amplification of a second circular polynucleotide hybridized tothe second capture probe (preferably, at the same time the firstamplified polynucleotide is produced). The second circularpolynucleotide comprises at least one copy of a polynucleotide sequenceidentical to a polynucleotide sequence in the second label probe, andthe second amplified polynucleotide thus comprises a plurality of copiesof a polynucleotide sequence complementary to the polynucleotidesequence in the second label probe. The plurality of second label probesis then hybridized to the second amplified polynucleotide. The amplifiedpolynucleotides remain associated (e.g., covalently) with the captureprobe(s), and the label probes are thus captured to the nucleic acidtargets. A circular polynucleotide can be provided and hybridized to thecapture probe, or a linear polynucleotide that is circularized byligation after it binds to the capture probe (e.g., a padlock probe) canbe employed. Techniques for rolling circle amplification, including useof padlock probes, are well known in the art. See, e.g., Larsson et al.(2004) “In situ genotyping individual DNA molecules by target-primedrolling-circle amplification of padlock probes” Nat Methods.1(3):227-32, Nilsson et al. (1994) Science 265:2085-2088, and Antson elal. (2000) “PCR-generated padlock probes detect single nucleotidevariation in genomic DNA” Nucl Acids Res 28(12):E58.

Potential capture probe sequences are optionally examined for possibleinteractions with non-corresponding nucleic acid targets, thepreamplifiers, the amplifiers, the label probes, and/or any relevantgenomic sequences, for example. Sequences expected to cross-hybridizewith undesired nucleic acids are typically not selected for use in thecapture probes. Examination can be, e.g., visual (e.g., visualexamination for complementarity), computational (e.g., a BLAST search ofthe relevant genomic database, or computation and comparison of bindingfree energies), and/or experimental (e.g., cross-hybridizationexperiments). Label probe sequences are preferably similarly examined,to help minimize potential undesirable cross-hybridization.

A capture probe, preamplifier, amplifier, and/or label probe optionallycomprises at least one non-natural nucleotide. For example, a captureprobe and a preamplifier (or amplifier or label probe) that hybridizesto it optionally comprise, at complementary positions, at least one pairof non-natural nucleotides that base pair with each other but that donot Watson-Crick base pair with the bases typical to biological DNA orRNA (i.e., A, C, G, T, or U). Examples of nonnatural nucleotidesinclude, but are not limited to, Locked Nucleic Acid™ nucleotides(available from Exiqon A/S, (www.) exiqon.com; see, e.g., SantaLucia Jr.(1998) Proc Natl Acad Sci 95:1460-1465) and isoG, isoC, and othernucleotides used in the AEGIS system (Artificially Expanded GeneticInformation System, available from EraGen Biosciences, (www.)eragen.com; see, e.g., U.S. Pat. Nos. 6,001,983, 6,037,120, and6,140,496). Use of such non-natural base pairs (e.g., isoG-isoC basepairs) in the probes can, for example, reduce background and/or simplifyprobe design by decreasing cross hybridization, or it can permit use ofshorter probes when the non-natural base pairs have higher bindingaffinities than do natural base pairs.

As noted, the methods are useful for multiplex detection of nucleicacids, including simultaneous detection of more than two nucleic acidtargets. Thus, the cell optionally comprises or is suspected ofcomprising a third nucleic acid target, and the methods optionallyinclude: providing a third label probe comprising a third label, whereina third signal from the third label is distinguishable from the firstand second signals, providing at least a third capture probe,hybridizing in the cell the third capture probe to the third nucleicacid target (when the third target is present in the cell), capturingthe third label probe to the third capture probe, and detecting thethird signal from the third label. Fourth, fifth, sixth, etc. nucleicacid targets are similarly simultaneously detected in the cell ifdesired.

A nucleic acid target can be essentially any nucleic acid that isdesirably detected in the cell. For example, a nucleic acid target canbe a DNA, a chromosomal DNA, an RNA, an mRNA, a microRNA, a ribosomalRNA, or the like. The nucleic acid target can be a nucleic acidendogenous to the cell. As another example, the target can be a nucleicacid introduced to or expressed in the cell by infection of the cellwith a pathogen, for example, a viral or bacterial genomic RNA or DNA, aplasmid, a viral or bacterial mRNA, or the like.

The first and second (and/or optional third, fourth, etc.) nucleic acidtargets can be part of a single nucleic acid molecule, or they can beseparate molecules. Various advantages and applications of bothapproaches are discussed in greater detail below and in the sectionentitled “Implementation, applications, and advantages.” In one class ofembodiments, the first nucleic acid target is a first mRNA and thesecond nucleic acid target is a second mRNA. In another class ofembodiments, the first nucleic acid target comprises a first region ofan mRNA and the second nucleic acid target comprises a second region ofthe same mRNA; this approach can increase specificity of detection ofthe mRNA. In another class of embodiments, the first nucleic acid targetcomprises a first chromosomal DNA polynucleotide sequence and the secondnucleic acid target comprises a second chromosomal DNA polynucleotidesequence. The first and second chromosomal DNA polynucleotide sequencesare optionally located on the same chromosome, e.g., within the samegene, or on different chromosomes.

In one aspect, the signal(s) from nucleic acid target(s) are normalized.In one class of embodiments, the second nucleic acid target comprises areference nucleic acid, and the method includes normalizing the firstsignal to the second signal. The reference nucleic acid is a nucleicacid selected as a standard of comparison. It will be evident thatchoice of the reference nucleic acid can depend on the desiredapplication. For example, for gene expression analysis, where the firstand optional third, fourth, etc. nucleic acid targets are mRNAs whoseexpression levels are to be determined, the reference nucleic acid canbe an mRNA transcribed from a housekeeping gene. As another example, thefirst nucleic acid target can be an mRNA whose expression is altered ina pathological state, e.g., an mRNA expressed in a tumor cell and not anormal cell or expressed at a higher level in a tumor cell than in anormal cell, while the second nucleic acid target is an mRNA expressedfrom a housekeeping gene or similar gene whose expression is not alteredin the pathological state. As yet another example, the first nucleicacid target can be a chromosomal DNA sequence that is amplified ordeleted in a tumor cell, while the second nucleic acid target is anotherchromosomal DNA sequence that is maintained at its normal copy number inthe tumor cell. Exemplary reference nucleic acids are described herein,and many more are well known in the art.

Optionally, results from the cell are compared with results from areference cell. That is, the first and second targets are also detectedin a reference cell, for example, a non-tumor, uninfected, or otherhealthy normal cell, chosen as a standard of comparison depending on thedesired application. The signals can be normalized to a referencenucleic acid as noted above. As just one example, the first nucleic acidtarget can be the Her-2 gene, with the goal of measuring Her-2 geneamplification. Signal from Her-2 can be normalized to that from areference gene, whose copy number is stably maintained in the genomicDMA. The normalized signal for the Her-2 gene from a target cell (e.g.,a tumor cell or suspected tumor cell) can be compared to the normalizedsignal from a reference cell (e.g., a normal cell), to determine copynumber in the cancer cell in comparison to normal cells.

The label (first, second, third, etc.) can be essentially any convenientlabel that directly or indirectly provides a detectable signal. In oneaspect, the first label is a first fluorescent label and the secondlabel is a second fluorescent label. Detecting the signal from thelabels thus comprises detecting fluorescent signals from the labels. Avariety of fluorescent labels whose signals can be distinguished fromeach other are known, including, e.g., fluorophores and quantum dots. Asother examples, the label can be a luminescent label, a light-scatteringlabel (e.g., colloidal gold particles), or an enzyme (e.g., alkalinephosphatase or horseradish peroxidase).

The methods can be used to detect the presence of the nucleic acidtargets in cells from essentially any type of sample. For example, thesample can be derived from a bodily fluid, a bodily waste, blood, bonemarrow, sputum, urine, lymph node, stool, vaginal secretions, cervicalpap smear, oral swab or other swab or smear, spinal fluid, saliva,sputum, ejaculatory fluid, semen, lymph fluid, an intercellular fluid, atissue (e.g., a tissue homogenate), a biopsy, and/or a tumor. The sampleand/or the cell can be derived from one or more of a human, an animal, aplant, and a cultured cell. Samples derived from even relatively largevolumes of materials such as bodily fluid or bodily waste can bescreened in the methods of the invention, and removal of such materialsis relatively non-invasive. Samples are optionally taken from a patient,following standard laboratory methods after informed consent.

The methods for detecting nucleic acid targets in cells can be used toidentify the cells. For example, a cell can be identified as being of adesired type based on which nucleic acids, and in what levels, itcontains. Thus, in one class of embodiments, the methods includeidentifying the cell as a desired target cell based on detection of thefirst and second signals (and optional third, fourth, etc. signals) fromwithin the cell. The cell can be identified on the basis of the presenceor absence of one or more of the nucleic acid targets. Similarly, thecell can be identified on the basis of the relative signal strength fromor expression level of one or more of the nucleic acid targets. Signalsare optionally normalized as noted above and/or compared to those from areference cell.

The methods can be applied to detection and identification of even rarecell types. Thus, the sample including the cell can be a mixture ofdesired target cells and other, nontarget cells, which can be present inexcess of the target cells. For example, the ratio of target cells tocells of all other type(s) in the sample is optionally less than1:1×10⁴, less than 1:1×10⁵, less than 1:1×10⁶, less than 1:1×10⁷, lessthan 1:1×10⁸, or even less than 1:1×10⁹.

Essentially any type of cell that can be differentiated based on itsnucleic acid content (presence, absence, expression level or copy numberof one or more nucleic acids) can be detected and identified using themethods and a suitable choice of nucleic acid targets. As just a fewexamples, the cell can be a circulating tumor cell, a virally infectedcell, a fetal cell in maternal blood, a bacterial cell or othermicroorganism in a biological sample (e.g., blood or other body fluid),an endothelial cell, precursor endothelial cell, or myocardial cell inblood, a stem cell, or a T-cell. Rare cell types can be enriched priorto performing the methods, if necessary, by methods known in the art(e.g., lysis of red blood cells, isolation of peripheral bloodmononuclear cells, further enrichment of rare target cells throughmagnetic-activated cell separation (MACS), etc.). The methods areoptionally combined with other techniques, such as DAPI staining fornuclear DNA. It will be evident that a variety of different types ofnucleic acid markers are optionally detected simultaneously by themethods and used to identify the cell. For example, a cell can beidentified based on the presence or relative expression level of onenucleic acid target in the cell and the absence of another nucleic acidtarget from the cell; e.g., a circulating tumor cell can be identifiedby the presence or level of one or more markers found in the tumor celland not found (or found at different levels) in blood cells, and itsidentity can be confirmed by the absence of one or more markers presentin blood cells and not circulating tumor cells. The principle may beextended to using any other type of markers such as protein basedmarkers in single cells.

The cell is typically fixed and permeabilized before hybridization ofthe capture probes, to retain the nucleic acid targets in the cell andto permit the capture probes, label probes, etc. to enter the cell. Thecell is optionally washed to remove materials not captured to one of thenucleic acid targets. The cell can be washed after any of various steps,for example, after hybridization of the capture probes to the nucleicacid targets to remove unbound capture probes, after hybridization ofthe preamplifiers, amplifiers, and/or label probes to the captureprobes, and/or the like.

The various capture and hybridization steps can be performedsimultaneously or sequentially, in essentially any convenient order.Preferably, a given hybridization step is accomplished for all of thenucleic acid targets at the same time. For example, all the captureprobes (first, second, etc.) can be added to the cell at once andpermitted to hybridize to their corresponding targets, the cell can bewashed, amplifiers (first, second, etc.) can be hybridized to thecorresponding capture probes, the cell can be washed, the label probes(first, second, etc.) can be hybridized to the corresponding amplifiers,and the cell can then be washed again prior to detection of the labels.As another example, the capture probes can be hybridized to the targets,the cell can be washed, amplifiers and label probes can be addedtogether and hybridized, and the cell can then be washed prior todetection. It will be evident that double-stranded nucleic acidtarget(s) are preferably denatured, e.g., by heat, prior tohybridization of the corresponding capture probe(s) to the target(s).

Preferably, the cell is in suspension for all or most of the steps ofthe method, for ease of handling. However, the methods are alsoapplicable to cells in solid tissue samples (e.g., tissue sections)and/or cells immobilized on a substrate (e.g., a slide or othersurface). Thus, in one class of embodiments, the cell is in suspensionin the sample comprising the cell, and/or the cell is in suspensionduring the hybridizing, capturing, and/or detecting steps. For example,the cell can be in suspension in the sample and during thehybridization, capture, optional washing, and detection steps. In otherembodiments, the cell is in suspension in the sample comprising thecell, and the cell is fixed on a substrate during the hybridizing,capturing, and/or detecting steps. For example, the cell can be insuspension during the hybridization, capture, and optional washing stepsand immobilized on a substrate during the detection step.

Signals from the labels can be detected, and their intensitiesoptionally measured, by any of a variety of techniques well known in theart. For example, in embodiments in which the cell is in suspension, thefirst and second (and optional third, etc.) signals can be convenientlydetected by flow cytometry. In embodiments in which cells areimmobilized on a substrate, the first and second (and optional thirdetc.) signals can be detected, for example, by laser scanner ormicroscope, e.g., a fluorescent or automated scanning microscope. Asnoted, detection is at the level of individual, single cells. Signalsfrom the labels are typically detected in a single operation (e.g., asingle flow cytometry run or a single microscopy or scanning session),rather than sequentially in separate operations for each label. Such asingle detection operation can, for example, involve changing opticalfilters between detection of the different labels, but it does notinvolve detection of the first label followed by capture of the secondlabel and then detection of the second label. In some embodiments, thefirst and second (and optional third etc.) labels are captured to theirrespective targets simultaneously but are detected in separate detectionsteps or operations.

Additional features described herein, e.g., in the section belowentitled “Implementation, applications, and advantages,” can be appliedto the methods, as relevant. For example, as described in greater detailbelow, a label probe can include more than one label, identical ordistinct. Signal strength is optionally adjusted between targetsdepending on their expected copy numbers, if desired; for example, thesignal for an mRNA expressed at low levels can be amplified to a greaterdegree (e.g., by use of more labels per label probe and/or use ofpreamplifiers and amplifiers to capture more label probes per copy ofthe target) than the signal for a highly expressed mRNA.

In another aspect of the invention, two or more nucleic acids aredetected by PCR amplification of the nucleic acids in situ in individualcells. To prevent leakage of the resulting amplicons out of the cells, awater-oil emulsion can be made as mentioned in Li et al. (2006) “BEAMingup for detection and quantification of rare sequence variants” NatureMethods 3(2):95-7 that separates single cells into differentcompartments.

Detection of Relative Levels by Normalization to Reference Nucleic Acids

As discussed briefly above, the signal detected for a nucleic acid ofinterest can be normalized to that of a standard, reference nucleicacid. One general class of embodiments thus provides methods of assayinga relative level of one or more target nucleic acids in an individualcell. In the methods, a sample comprising the cell is provided. The cellcomprises or is suspected of comprising a first, target nucleic acid,and it comprises a second, reference nucleic acid. A first label probecomprising a first label and a second label probe comprising a secondlabel, wherein a first signal from the first label is distinguishablefrom a second signal from the second label, are also provided. In thecell, the first label probe is captured to the first, target nucleicacid (when the first, target nucleic acid is present in the cell) andthe second label probe is captured to the second, reference nucleicacid. The first signal from the first label and the second signal fromthe second label are then detected in the individual cell, and theintensity of each signal is measured. The intensity of the first signalis normalized to the intensity of the second (reference) signal. Thelevel of the first, target nucleic acid relative to the level of thesecond, reference nucleic acid in the cell is thereby assayed, since thefirst and second labels are associated with their respective nucleicacids. The methods are optionally quantitative, permitting measurementof the amount of the first, target nucleic acid relative to the amountof the second, reference nucleic acid in the cell. Thus, the intensityof the first signal normalized to that of the second signal can becorrelated with a quantity of the first, target nucleic acid present inthe cell.

The label probes can bind directly to the nucleic acids. For example,the first label probe can hybridize to the first, target nucleic acidand/or the second label probe can hybridize to the second, referencenucleic acid. Alternatively, some or all of the label probes can beindirectly bound to their corresponding nucleic acids, e.g., throughcapture probes. For example, the first and second label probes can binddirectly to the nucleic acids, or one can bind directly while the otherbinds indirectly, or both can bind indirectly.

The label probes are optionally captured to the nucleic acids viacapture probes. In one class of embodiments, at least a first captureprobe and at least a second capture probe are provided. In the cell, thefirst capture probe is hybridized to the first, target nucleic acid andthe second capture probe is hybridized to the second, reference nucleicacid. The first label probe is captured to the first capture probe andthe second label probe is captured to the second capture probe, therebycapturing the first label probe to the first, target nucleic acid andthe second label probe to the second, reference nucleic acid. Thefeatures described for the methods above apply to these embodiments aswell, with respect to configuration and number of the label and captureprobes, optional use of preamplifiers and/or amplifiers, rolling circleamplification of circular polynucleotides, and the like.

The methods can be used for multiplex detection of nucleic acids,including simultaneous detection of two or more target nucleic acids.Thus, the cell optionally comprises or is suspected of comprising athird, target nucleic acid, and the methods optionally include:providing a third label probe composing a third label, wherein a thirdsignal from the third label is distinguishable from the first and secondsignals; capturing, in the cell, the third label probe to the third,target nucleic acid (when present in the cell); detecting the thirdsignal from the third label, which detecting comprises measuring anintensity of the third signal; and normalizing the intensity of thethird signal to the intensity of the second signal. Alternatively, thethird signal can be normalized to that from a different referencenucleic acid. Fourth, fifth, sixth, etc. nucleic acids are similarlysimultaneously detected in the cell if desired. The third, fourth,fifth, etc. label probes are optionally hybridized directly to theircorresponding nucleic acid, or they can be captured indirectly viacapture probes as described for the first and second label probes.

The methods can be used for gene expression analysis, detection of geneamplification or deletion, or detection or diagnosis of disease, as justa few examples. A target nucleic acid can be essentially any nucleicacid that is desirably detected in the cell. For example, a targetnucleic acid can be a DNA, a chromosomal DNA, an RNA, an mRNA, amicroRNA, a ribosomal RNA, or the like. The target nucleic acid can be anucleic acid endogenous to the cell, or as another example, the targetcan be a nucleic acid introduced to or expressed in the cell byinfection of the cell with a pathogen, for example, a viral or bacterialgenomic RNA or DNA, a plasmid, a viral or bacterial mRNA, or the like.The reference nucleic acid can similarly be a DNA, an mRNA, achromosomal DNA, an mRNA, an RNA endogenous to the cell, or the like.

As described above, choice of the reference nucleic acid can depend onthe desired application. For example, for gene expression analysis,where the first and optional third, fourth, etc. target nucleic acidsare mRNAs whose expression levels are to be determined, the referencenucleic acid can be an mRNA transcribed from a housekeeping gene. Asanother example, the first, target nucleic acid can be an mRNA whoseexpression is altered in a pathological state, e.g., an mRNA expressedin a tumor cell and not a normal cell or expressed at a higher level ina tumor cell than in a normal cell, while the reference nucleic acid isan mRNA expressed from a housekeeping gene or similar gene whoseexpression is not altered in the pathological state. In a similarexample, the target nucleic acid can be a viral or bacterial nucleicacid while the reference nucleic acid is endogenous to the cell. As yetanother example, the first, target nucleic acid can be a chromosomal DNAsequence that is amplified or deleted in a tumor cell, while thereference nucleic acid is another chromosomal DNA sequence that ismaintained at its normal copy number in the tumor cell. Exemplaryreference nucleic acids are described herein, and many more are wellknown in the art.

In one class of embodiments, the first, target nucleic acid is a firstmRNA and the second, reference nucleic acid is a second mRNA. In anotherclass of embodiments, the first, target nucleic acid comprises a firstchromosomal DNA polynucleotide sequence and the second, referencenucleic acid comprises a second chromosomal DNA polynucleotide sequence.The first and second chromosomal DNA polynucleotide sequences areoptionally located on the same chromosome or on different chromosomes.

Optionally, normalized results from the cell are compared withnormalized results from a reference cell. That is, the target andreference nucleic acids are also detected in a reference cell, forexample, a non-tumor, uninfected, or other healthy normal cell, chosenas a standard of comparison depending on the desired application. Asjust one example, the first, target nucleic acid can be the Her-2 gene,with the goal of measuring Her-2 gene amplification. Signal from Her-2can be normalized to that from a reference gene whose copy number isstably maintained in the genomic DNA. The normalized signal for theHer-2 gene from a target cell (e.g., a tumor cell or suspected tumorcell) can be compared to the normalized signal from a reference cell(e.g., a normal cell), to determine copy number in the cancer cell incomparison to normal cells.

Signal strength is optionally adjusted between the target and referencenucleic acids depending on their expected copy numbers, if desired. Forexample, the signal for a target mRNA expressed at low levels can beamplified to a greater degree (e.g., by use of more labels per labelprobe and/or use of capture probes, preamplifiers and amplifiers tocapture more label probes per copy of the target) than the signal for ahighly expressed mRNA (which can, e.g., be detected by direct binding ofthe label probe to the reference nucleic acid, by use of capture probesand amplifier without a preamplifier, or the like).

The methods for assaying relative levels of target nucleic acids incells can be used to identify the cells. For example, a cell can beidentified as being of a desired type based on which nucleic acids, andin what levels, it contains. Thus, in one class of embodiments, themethods include identifying the cell as a desired target cell based onthe normalized first signal (and optional normalized third, fourth, etc.signals). As described herein, the cell can be identified on the basisof the presence or absence of one or more of the target nucleic acids.Similarly, the cell can be identified on the basis of the relativesignal strength from or expression level of one or more target nucleicacids. Signals are optionally compared to those from a reference cell.

The methods can be applied to detection and identification of even rarecell types. Thus, the sample including the cell can be a mixture ofdesired target cells and other, nontarget cells, which can be present inexcess of the target cells. For example, the ratio of target cells tocells of all other type(s) in the sample is optionally less than1:1×10⁴, less than 1:1×10⁵, less than 1:1×10⁶, less than 1:1×10⁷, lessthan 1:1×10⁸, or even less than 1:1×10⁹.

Essentially any type of cell that can be differentiated based on itsnucleic acid content (presence, absence, or copy number of one or morenucleic acids) can be detected and identified using the methods and asuitable choice of target and reference nucleic acids. As just a fewexamples, the cell can be a circulating tumor cell, a virally infectedcell, a fetal cell in maternal blood, a bacterial cell or othermicroorganism in a biological sample (e.g., blood or other body fluid),or an endothelial cell, precursor endothelial cell, or myocardial cellin blood. Rare cell types can be enriched prior to performing themethods, if necessary, by methods known in the art (e.g., lysis of redblood cells, isolation of peripheral blood mononuclear cells, etc.). Themethods are optionally combined with other techniques, such as DAPIstaining for nuclear DNA. It will be evident that a variety of differenttypes of nucleic acid markers are optionally detected simultaneously bythe methods and used to identify the cell. For example, a cell can beidentified based on the presence or relative expression level of onetarget nucleic acid in the cell and the absence of another targetnucleic acid from the cell; e.g., a circulating tumor cell can beidentified by the presence or level of one or more markers found in thetumor cell and not found (or found at different levels) in blood cells,and by the absence of one or more markers present in blood cells and notcirculating tumor cells. The principle may be extended to using anyother type of markers such as protein based markers in single cells.

Essentially all of the features noted for the methods above apply tothese embodiments as well, as relevant; for example, with respect tosource of sample, fixation and permeabilization of the cell, washing thecell, denaturation of double-stranded target and reference nucleicacids, type of labels, use of optional blocking probes, detection ofsignals, detection (and intensity measurement) by flow cytometry ormicroscopy, presence of the cell in suspension or immobilized on asubstrate, and/or the like. Also, additional features described herein,e.g., in the section entitled “Implementation, applications, andadvantages” can be applied to the methods, as relevant.

The methods of the invention can be used for gene expression analysis insingle cells. Currently, gene expression analysis deals withheterogeneous cell populations such as blood or tumor specimens. Bloodcontains various subtypes of leukocytes, and when changes in geneexpression of whole blood or RNA isolated from blood are measured, it isnot known what subtype of blood cells actually changed their geneexpression. It is possible that gene expression of only a certainsubtype of blood cells is affected in a disease state or by drugtreatment, for example. Technology that can measure gene expression insingle cells, so changes of gene expression in single cells can beexamined, is thus desirable. Similarly, a tumor specimen contains aheterogeneous cell population including tumor cells, normal cells,stromal cells, immune cells, etc. Current technology looks at the sum ofthe expression of all those cells through total RNA or cell lysate.However, the overall expression change may not be representative of thatin target tumor cells. So again, it would be useful to look at theexpression changes in single cells so that the target tumor cells can beexamined specifically, to see how the target cells change in geneexpression and how they respond to drug treatment, for example.

In one aspect, the present invention provides methods for geneexpression analysis in single cells. Single cell gene expressionanalysis can be accomplished by measuring expression of a target geneand normalizing against the expression of a housekeeping gene, asdescribed above. As just a couple of examples, the normalized expressionin a disease state can be compared to that in the normal state, or theexpression in a drug treated state can be compared to that in the normalstate. The change of expression level in single cells may havebiological significance indicating disease progression, drug therapeuticefficacy and/or toxicity, tumor staging and classification, etc.

Accordingly, one general class of embodiments provides methods ofperforming comparative gene expression analysis in single cells. In themethods, a first mixed cell population comprising one or more cells of aspecified type is provided. A second mixed cell population comprisingone or more cells of the specified type is also provided. An expressionlevel of one or more target nucleic acids relative to a referencenucleic acid is measured in the cells of the specified type of the firstpopulation, to provide a first expression profile. An expression levelof the one or more target nucleic acids relative to the referencenucleic acid is measured in the cells of the specified type of thesecond population, to provide a second expression profile. The first andsecond expression profiles are compared.

In one class of embodiments, the one or more target nucleic acids areone or more mRNAs, e.g., two or more, three or more, four or more, etc.mRNAs. The expression level of each mRNA can be determined relative tothat of a housekeeping gene whose mRNA serves as the reference nucleicacid.

The first and/or second mixed cell population contains at least oneother type of cell in addition to the specified type, more typically atleast two or more other types of cells, and optionally several to manyother types of cells (e.g., as is found in whole blood, a tumor, orother complex biological sample). The ratio of cells of the specifiedtype to cells of all other type(s) in the first or second mixed cellpopulation is optionally less than 1:1×10⁴, less than 1:1×10⁵, less than1:1×10⁶, less than 1:1×10⁷, less than 1:1×10⁸, or even less than1:1×10⁹.

As will be evident, a change in gene expression profile between the twopopulations may indicate a disease state or progression, a drugresponse, a therapeutic efficacy, etc. Thus, for example, the firstmixed cell population can be from a patient who has been diagnosed orwho is to be diagnosed with a particular disease or disorder, while thesecond mixed population is from a healthy individual. Similarly, thefirst and second mixed populations can be from a single individual buttaken at different time points, for example, to follow diseaseprogression or to assess response to drug treatment. Accordingly, thefirst mixed cell population can be taken from an individual (e.g., ahuman) before treatment is initiated with a drug or other compound,while the second population is taken at a specified time after treatmentis initiated. As another example, the first mixed population can be froma treated individual while the second mixed population is from anuntreated individual.

Essentially all of the features noted for the methods above apply tothese embodiments as well, as relevant; for example, with respect totype of target and reference nucleic acids, cell type, source of sample,fixation and permeabilization of the cell, washing the cell,denaturation of double-stranded target and reference nucleic acids, typeof labels, use and configuration of label probes, capture probes,preamplifiers and/or amplifiers, use of optional blocking probes,detection of signals, detection (and intensity measurement) by flowcytometry or microscopy, presence of the cell in suspension orimmobilized on a substrate, and/or the like. Exemplary target andreference nucleic acids are described herein.

In another aspect, the methods can be used to compare copy number insingle cells from a first population (e.g., tumor cells) with copynumber in single cells from a second population (e.g., normal cells usedas a reference). The nucleic acid target(s) can be transcripts orgenomic DNA, where, for example, the degree of amplification or deletionof genes such as her-2 can correlate with tumor progression. In anotheraspect, the methods can be applied to gene expression analysis in singlecells in even a single population, including, for example, cells of thesame type but at different stages of the cell cycle.

Label Density

The methods of the invention permit far more labels to be captured tosmall regions of target nucleic acids than do currently existingtechniques. For example, standard FISH techniques typically use probesthat cover 20 kb or more, and a probe typically has fluorophoreschemically conjugated at a density of approximately one fluorescentmolecule per seven nucleotides of the probe. When molecular beacontarget detection is employed, one label pair is captured to the targetin the region covered by the beacon, typically about 40 nucleotides. Foradditional discussion of exemplary current techniques, see, e.g., U.S.patent application publications 2004/0091880 and 2005/0181463, U.S. Pat.No. 6,645,731, and international patent application publications WO95/09245 and 03/019141.

Methods described herein, in comparison, readily permit capture ofhundreds of labels (e.g., 400 or more) to the region of the targetcovered by a single capture probe, e.g., 20-25 nucleotides or more. Thetheoretical degree of amplification achieved from a single capture probeis readily calculated for any given configuration of capture probes,amplifiers, etc; for example, the theoretical degree of amplificationachieved from a single capture probe, and thus the number of labels perlength in nucleotides of the capture probe, can be equal to the numberof preamplifiers bound to the capture probe times the number ofamplifiers that bind each preamplifier times the number of label probesthat bind each preamplifier limes the number of labels per label probe.

Thus, in one aspect, the invention provides methods that facilitateassociation of a high density of labels to target nucleic acids incells. One general class of embodiments provides methods of detectingtwo or more nucleic acid targets in an individual cell. In the methods,a sample comprising the cell is provided. The cell comprises or issuspected of comprising a first nucleic acid target and a second nucleicacid target. In the cell, a first label is captured to the first nucleicacid target (when present in the cell) and a second label is captured tothe second nucleic acid target (when present in the cell). A firstsignal from the first label is distinguishable from a second signal fromthe second label. As noted, the labels are captured at high density.Thus, an average of at least one copy of the first label per nucleotideof the first nucleic acid target is captured to the first nucleic acidtarget over a region that spans at least 20 contiguous nucleotides ofthe first nucleic acid target, and an average of at least one copy ofthe second label per nucleotide of the second nucleic acid target iscaptured to the second nucleic acid target over a region that spans atleast 20 contiguous nucleotides of the second nucleic acid target. Thefirst signal from the first label and the second signal from the secondlabel are detected.

In one class of embodiments, an average of at least four, eight, ortwelve copies of the first label per nucleotide of the first nucleicacid target are captured to the first nucleic acid target over a regionthat spans at least 20 contiguous nucleotides of the first nucleic acidtarget, and an average of at least four, eight, or twelve copies of thesecond label per nucleotide of the second nucleic acid target arecaptured to the second nucleic acid target over a region that spans atleast 20 contiguous nucleotides of the second nucleic acid target. Inone embodiment, an average of at least sixteen copies of the first labelper nucleotide of the first nucleic acid target are captured to thefirst nucleic acid target over a region that spans at least 20contiguous nucleotides of the first nucleic acid target, and an averageof at least sixteen copies of the second label per nucleotide of thesecond nucleic acid target are captured to the second nucleic acidtarget over a region that spans at least 20 contiguous nucleotides ofthe second nucleic acid target.

Essentially all of the features noted for the methods above apply tothese embodiments as well, as relevant, for example, with respect totype, of labels, detection of signals, type, treatment, and suspensionof the cell, and/or the like. The regions of the first and secondnucleic acid targets optionally span at least 25, 50, 100, 200, or morecontiguous nucleotides and/or at most 2000, 1000, 500, 200, 100, 50, orfewer nucleotides. A like density of third, fourth, fifth, sixth, etc.labels is optionally present for (e.g., captured to) third, fourth,fifth, sixth, etc. nucleic acid targets.

Detection of Target Cells

As described above, cells can be detected and identified by detectingtheir constituent nucleic acids. For certain applications, for example,detection of rare cells from large heterogeneous mixtures of cells,detection of multiple, redundant nucleic acid markers in order to detectthe rare cell is advantageous. The following hypothetical exampleillustrates one advantage of detecting redundant markers.

Say that circulating tumor cells (CTC) are to be detected from a bloodsample in which the CTC concentration is one in 10⁶ normal white bloodcells. If a single nucleic acid marker for the CTC (e.g., a nucleic acidwhose presence or copy number can uniquely and sufficiently distinguishthe cell from the rest of the cell population) has a detectionspecificity of 1 in 10³, 1000 cells will be mistakenly identified as“CTC” when 10⁶ cells are counted. (Such false positives can result fromrandom background signal generated by nonspecific binding of therelevant probe(s) or from similar factors.) If an additional independentmarker is included which, on its own, also has a detection specificityof 1 in 10³, and if a cell is identified as a CTC only if both markersare positive, the combined detection specificity is now theoreticallydramatically increased, to 1 in 10³×10³=10⁶. This specificity issufficient for direct CTC detection in normal white blood cells underthese assumptions. Similarly, if three independent redundant markers areused for identification of CTC, the detection specificity can be boostedto 1 in 10⁹. Use of two or more redundant markers thus reduces thenumber of false positives and facilitates detection of even rare cellsfrom complex samples.

Accordingly, one general class of embodiments provides methods ofdetecting an individual cell of a specified type. In the methods, asample comprising a mixture of cell types including at least one cell ofthe specified type is provided. A first label probe comprising a firstlabel and a second label probe comprising a second label, wherein afirst signal from the first label is distinguishable from a secondsignal from the second label, are provided. In the cell, the first labelprobe is captured to a first nucleic acid target (when the first nucleicacid target is present in the cell) and the second label probe iscaptured to a second nucleic acid target (when the second nucleic acidtarget is present in the cell). The first signal from the first labeland the second signal from the second label are detected and correlatedwith the presence, absence, or amount of the corresponding, first andsecond nucleic acid targets in the cell. The cell is identified as beingof the specified type based on detection of the presence, absence, oramount (e.g., a non-zero amount) of both the first and second nucleicacid targets within the cell, where the specified type of cell isdistinguishable from the other cell type(s) in the mixture on the basisof either the presence, absence, or amount of the first nucleic acidtarget or the presence, absence, or amount of the second nucleic acidtarget in the cell (that is, the nucleic acid targets are redundantmarkers for the specified cell type). An intensity of the first signaland an intensity of the second signal are optionally measured andcorrelated with a quantity of the corresponding nucleic acid present inthe cell.

Each nucleic acid target that serves as a marker for the specified celltype can distinguish the cell type by its presence in the cell, by itsamount (copy number, e.g., its genomic copy number or its transcriptexpression level), or by its absence from the cell (a negative marker).A set of nucleic acid targets can include different types of suchmarkers; that is, one nucleic acid target can serve as a positivemarker, distinguishing the cell by its presence or non-zero amount inthe cell, while another serves as a negative marker, distinguishing thecell by its absence from the cell. For example, in one class ofembodiments, the cell comprises a first nucleic acid target and a secondnucleic acid target, and the cell is identified as being of thespecified type based on detection of the presence or amount of both thefirst and second nucleic acid targets within the cell, where thespecified type of cell is distinguishable from the other cell type(s) inthe mixture on the basis of either the presence or amount of the firstnucleic acid target or the presence or amount of the second nucleic acidtarget in the cell.

The label probes can bind directly to the nucleic acid targets. Forexample, the first label probe can hybridize to the first nucleic acidtarget and/or the second label probe can hybridize to the second nucleicacid target. Alternatively, some or all of the label probes can beindirectly bound to their corresponding nucleic acid targets, e.g.,through capture probes. For example, the first and second label probescan bind directly to the nucleic acid targets, or one can bind directlywhile the other binds indirectly, or both can bind indirectly.

The label probes are optionally captured to the nucleic acid targets viacapture probes. In one class of embodiments, at least a first captureprobe and at least a second capture probe are provided. In the cell, thefirst capture probe is hybridized to the first nucleic acid target andthe second capture probe is hybridized to the second nucleic acidtarget. The first label probe is captured to the first capture probe andthe second label probe is captured to the second capture probe, therebycapturing the first label probe to the first nucleic acid target and thesecond label probe to the second nucleic acid target. The featuresdescribed for the methods above apply to these embodiments as well, withrespect to configuration and number of the label and capture probes,optional use of preamplifiers and/or amplifiers, rolling circleamplification of circular polynucleotides, and the like.

Third, fourth, fifth, etc. nucleic acid targets are optionally detectedin the cell. For example, the method optionally includes: providing athird label probe comprising a third label, wherein a third signal fromthe third label is distinguishable from the first and second signals,capturing in the cell the third label probe to a third nucleic acidtarget (when present in the cell), and detecting the third signal fromthe third label. The third, fourth, fifth, etc. label probes areoptionally hybridized directly to their corresponding nucleic acid, orthey can be captured indirectly via capture probes as described for thefirst and second label probes.

The additional markers can be used in any of a variety of ways. Forexample, the cell can comprise the third nucleic acid target, and thefirst and/or second signal can be normalized to the third signal. Themethods can include identifying the cell as being of the specified typebased on the normalized first and/or second signal, e.g., in embodimentsin which the target cell type is distinguishable from the other celltype(s) in the mixture based on the copy number of the first and/orsecond nucleic acid targets, rather than purely on their presence in thetarget cell type and not in the other cell type(s). Examples includecells detectable based on a pattern of differential gene expression, CTCor other tumor cells detectable by overexpression of one or morespecific mRNAs, and CTC or other tumor cells detectable by amplificationor deletion of one or more specific chromosomal regions.

As another example, the third nucleic acid target can serve as a thirdredundant marker for the target cell type, e.g., to improve specificityof the assay for the desired cell type. Thus, in one class ofembodiments, the methods include correlating the third signal detectedfrom the cell with the presence, absence, or amount of the third nucleicacid target in the cell, and identifying the cell as being of thespecified type based on detection of the presence, absence, or amount ofthe first, second, and third nucleic acid targets within the cell,wherein the specified type of cell is distinguishable from the othercell type(s) in the mixture on the basis of either presence, absence, oramount of the first nucleic acid target, presence, absence, or amount ofthe second nucleic acid target, or presence, absence, or amount of thethird nucleic acid target in the cell.

As yet another example, the additional markers can assist in identifyingthe cell type. For example, the presence, absence, or amount of thefirst and third markers may suffice to identify the cell type, as couldthe presence, absence, or amount of the second and fourth markers; allfour markers could be detected to provide two redundant sets of markersand therefore increased specificity of detection. As another example,one or more additional markers can be used in negative selection againstundesired cell types; for example, identity of a cell as a CTC can befurther verified by the absence from the cell of one or more markerspresent in blood cells and not circulating tumor cells.

Detection of additional nucleic acid targets can also provide furtherinformation useful in diagnosis, outcome prediction or the like,regardless of whether the targets serve as markers for the particularcell type. For example, additional nucleic acid targets can includemarkers for proliferating potential, apoptosis, or other metastatic,genetic, or epigenetic changes.

Signals from the additional targets are optionally normalized to areference nucleic acid as described above. Signal strength is optionallyadjusted between targets depending on their expected copy numbers, ifdesired. Signals from the target nucleic acids in the cell areoptionally compared to those from a reference cell, as noted above.

A nucleic acid target can be essentially any nucleic acid that isdesirably detected in the cell. For example, a nucleic acid target canbe a DNA, a chromosomal DNA, an RNA, an mRNA, a microRNA, a ribosomalRNA, or the like. The nucleic acid target can be a nucleic acidendogenous to the cell. As another example, the target can be a nucleicacid introduced to or expressed in the cell by infection of the cellwith a pathogen, for example, a viral or bacterial genomic RNA or DNA, aplasmid, a viral or bacterial mRNA, or the like.

The first and second (and/or optional third, fourth, etc.) nucleic acidtargets can be part of a single nucleic acid molecule, or they can beseparate molecules. Various advantages and applications of bothapproaches are discussed in greater detail below, e.g., in the sectionentitled “Implementation, applications, and advantages.” In one class ofembodiments, the first nucleic acid target is a first mRNA and thesecond nucleic acid target is a second mRNA. In another class ofembodiments, the first nucleic acid target comprises a first region ofan mRNA and the second nucleic acid target comprises a second region ofthe same mRNA. In another class of embodiments, the first nucleic acidtarget comprises a first chromosomal DNA polynucleotide sequence and thesecond nucleic acid target comprises a second chromosomal DNApolynucleotide sequence. The first and second chromosomal DNApolynucleotide sequences are optionally located on the same chromosome,e.g., within the same gene, or on different chromosomes.

The methods can be applied to detection and identification of even rarecell types. For example, the ratio of cells of the specified type tocells of all other type(s) in the mixture is optionally less than1:1×10⁴, less than 1:1×10⁵, less than 1:1×10⁶, less than 1:1×10⁷, lessthan 1:1×10⁸, or even less than 1:1×10⁹.

Essentially any type of cell that can be differentiated based onsuitable markers (or redundant regions of a single marker, e.g., asingle mRNA or amplified/deleted chromosomal region) can be detected andidentified using the methods. As just a few examples, the cell can be acirculating tumor cell, a virally infected cell, a fetal cell inmaternal blood, a bacterial cell or other microorganism in a biologicalsample (e.g., blood or other body fluid), an endothelial cell, precursorendothelial cell, or myocardial cell in blood, stem cell, or T-cell.Rare cell types can be enriched prior to performing the methods, ifnecessary, by methods known in the art (e.g., lysis of red blood cells,isolation of peripheral blood mononuclear cells, etc.).

Essentially all of the features noted for the methods above apply tothese embodiments as well, as relevant; for example, with respect tosource of sample, fixation and permeabilization of the cell, washing thecell, denaturation of double-stranded nucleic acids, type of labels, useof optional blocking probes, detection of signals, detection (andintensity measurement) of signals from the individual cell by flowcytometry or microscopy, presence of the cell in suspension orimmobilized on a substrate, and/or the like. Also, additional featuresdescribed herein, e.g., in the section entitled “Implementation,applications, and advantages,” can be applied to the methods, asrelevant.

In another aspect, detection of individual cells of a specified type isperformed as described above, but the first and second nucleic acidtargets need not be redundant markers for that cell type. The nucleicacid targets can be essentially any desired nucleic acids, including,for example, redundant and/or non-redundant markers for the cell type.

Compositions and Kits

The invention also provides compositions useful in practicing orproduced by the methods. One exemplary class of embodiments provides acomposition that includes a fixed and permeabilized cell, which cellcomprises or is suspected of comprising a first nucleic acid target anda second nucleic acid target, at least a first capture probe capable ofhybridizing to the first nucleic acid target, at least a second captureprobe capable of hybridizing to the second nucleic acid target, a firstlabel probe comprising a first label, and a second label probecomprising a second label. A first signal from the first label isdistinguishable from a second signal from the second label. The celloptionally comprises the first and second capture probes and labelprobes. The first and second capture probes are optionally hybridized totheir respective nucleic acid targets in the cell.

The features described for the methods above for indirect capture of thelabel probes to the nucleic acid targets apply to these embodiments aswell. For example, the label probes can hybridize to the capture probes.In one class of embodiments, the composition includes a single firstcapture probe and a single second capture probe, where the first labelprobe is capable of hybridizing to the first capture probe and thesecond label probe is capable of hybridizing to the second captureprobe. In another class of embodiments, the composition includes two ormore first capture probes, two or more second capture probes, aplurality of the first label probes, and a plurality of the second labelprobes. A single first label probe is capable of hybridizing to each ofthe first capture probes, and a single second label probe is capable ofhybridizing to each of the second capture probes.

In another aspect, amplifiers can be employed to increase the number oflabel probes captured to each target. For example, in one class ofembodiments, the composition includes a single first capture probe, asingle second capture probe, a plurality of the first label probes, aplurality of the second label probes, a first amplifier, and a secondamplifier. The first amplifier is capable of hybridizing to the firstcapture probe and to the plurality of first label probes, and the secondamplifier is capable of hybridizing to the second capture probe and tothe plurality of second label probes. In another class of embodiments,the composition includes two or more first capture probes, two or moresecond capture probes, a multiplicity of the first label probes, amultiplicity of the second label probes, a first amplifier, and a secondamplifier. The first amplifier is capable of hybridizing to one of thefirst capture probes and to a plurality of first label probes, and thesecond amplifier is capable of hybridizing to one of the second captureprobes and to a plurality of second label probes.

In another aspect, preamplifiers and amplifiers are employed to capturethe label probes to the targets. In one class of embodiments, thecomposition includes a single first capture probe, a single secondcapture probe, a multiplicity of the first label probes, a multiplicityof the second label probes, a plurality of first amplifiers, a pluralityof second amplifiers, a first preamplifier, and a second preamplifier.The first preamplifier is capable of hybridizing to the first captureprobe and to the plurality of first amplifiers, and the secondpreamplifier is capable of hybridizing to the second capture probe andto the plurality of second amplifiers. The first amplifier is capable ofhybridizing to the first preamplifier and to a plurality of first labelprobes, and the second amplifier is capable of hybridizing to the secondpreamplifier and to a plurality of second label probes. In a relatedclass of embodiments, the composition includes two or more first captureprobes, two or more second capture probes, a multiplicity of the firstlabel probes, a multiplicity of the second label probes, a multiplicityof first amplifiers, a multiplicity of second amplifiers, a plurality offirst preamplifiers, and a plurality of second preamplifiers. The firstpreamplifier is capable of hybridizing to one of the first captureprobes and to a plurality of first amplifiers, the second preamplifieris capable of hybridizing to one of the second capture probes and to aplurality of second amplifiers, the first amplifier is capable ofhybridizing to the first preamplifier and to a plurality of first labelprobes, and the second amplifier is capable of hybridizing to the secondpreamplifier and to a plurality of second label probes. Optionally,additional preamplifiers can be used as intermediates between apreamplifier hybridized to the capture probe(s) and the amplifiers.

In the above classes of embodiments, one capture probe hybridizes toeach label probe, amplifier, or preamplifier. In alternative classes ofrelated embodiments, two or more capture probes hybridize to the labelprobe, amplifier, or preamplifier.

In one class of embodiments, the composition comprises a plurality ofthe first label probes, a plurality of the second label probes, a firstamplified polynucleotide produced by rolling circle amplification of afirst circular polynucleotide hybridized to the first capture probe, anda second amplified polynucleotide produced by rolling circleamplification of a second circular polynucleotide hybridized to thesecond capture probe. The first circular polynucleotide comprises atleast one copy of a polynucleotide sequence identical to apolynucleotide sequence in the first label probe, and the firstamplified polynucleotide comprises a plurality of copies of apolynucleotide sequence complementary to the polynucleotide sequence inthe first label probe (and can thus hybridize to a plurality of thelabel probes). The second circular polynucleotide comprises at least onecopy of a polynucleotide sequence identical to a polynucleotide sequencein the second label probe, and the second amplified polynucleotidecomprises a plurality of copies of a polynucleotide sequencecomplementary to the polynucleotide sequence in the second label probe.The composition can also include reagents necessary for producing theamplified polynucleotides, for example, an exogenously supplied nucleicacid polymerase, an exogenously supplied nucleic acid ligase, and/orexogenously supplied nucleoside triphosphates (e.g., dNTPs).

The cell optionally includes additional nucleic acid targets, and thecomposition (and cell) can include reagents for detecting these targets.For example, the cell can comprise or be suspected of comprising a thirdnucleic acid target, and the composition can include at least a thirdcapture probe capable of hybridizing to the third nucleic acid targetand a third label probe comprising a third label. A third signal fromthe third label is distinguishable from the first and second signals.The cell optionally includes fourth, fifth, sixth, etc. nucleic acidtargets, and the composition optionally includes fourth, fifth, sixth,etc. label probes and capture probes.

Essentially all of the features noted for the methods above apply tothese embodiments as well, as relevant; for example, with respect totype of nucleic acid target, location of various targets on a singlemolecule or on different molecules, type of labels, inclusion ofoptional blocking probes, and/or the like. For example, it is worthnoting that the second nucleic acid target optionally comprises areference nucleic acid. In other embodiments, the first and secondnucleic acid targets serve as markers for a specified cell type, e.g.,redundant markers.

The cell can be essentially any type of cell from any source,particularly a cell that can be differentiated based on its nucleic acidcontent (presence, absence, or copy number of one or more nucleicacids). As just a few examples, the cell can be a circulating tumorcell, a virally infected cell, a fetal cell in maternal blood, abacterial cell or other microorganism in a biological sample (e.g.,blood or other body fluid), or an endothelial cell, precursorendothelial cell, or myocardial cell in blood. For example, the cell canbe derived from a bodily fluid, blood, bone marrow, sputum, urine, lymphnode, stool, cervical pap smear, oral swab or other swab or smear,spinal fluid, saliva, sputum, semen, lymph fluid, an intercellularfluid, a tissue (e.g., a tissue homogenate), a biopsy, and/or a tumor.The cell can be derived from one or more, of a human, an animal, aplant, and a cultured cell.

The cell can be present in a mixture of cells, for example, a complexheterogeneous mixture. In one class of embodiments, the cell is of aspecified type, and the composition comprises one or more other types ofcells. These other cells can be present in excess, even large excess, ofthe cell. For example, the ratio of cells of the specified type to cellsof all other type(s) in the composition is optionally less than 1:1×10⁴,less than 1:1×10⁵, less than 1:1×10⁶, less than 1:1×10⁷, less than1:1×10⁸, or even less than 1:1×10⁹.

The cell is optionally immobilized on a substrate, present in a tissuesection, or the like. Preferably, however, the cell is in suspension inthe composition. The composition can be contained in a flow cytometer orsimilar instrument. Additional features described herein, e.g., in thesection entitled “implementation, applications, and advantages,” can beapplied to the compositions, as relevant.

Another aspect of the invention provides compositions in which a largenumber of labels are correlated with each target nucleic acid. Onegeneral class of embodiments thus provides a composition comprising acell, which cell includes a first nucleic acid target, a second nucleicacid target, a first label whose presence in the cell is indicative ofthe presence of the first nucleic acid target in the cell, and a secondlabel whose presence in the cell is indicative of the presence of thesecond nucleic acid target in the cell, wherein a first signal from thefirst label is distinguishable from a second signal from the secondlabel. An average of at least one copy of the first label is present inthe cell per nucleotide of the first nucleic acid target over a regionthat spans at least 20 contiguous nucleotides of the first nucleic acidtarget, and an average of at least one copy of the second label ispresent in the cell per nucleotide of the second nucleic acid targetover a region that spans at least 20 contiguous nucleotides of thesecond nucleic acid target.

In one class of embodiments, the copies of the first label arephysically associated with the first nucleic acid target, and the copiesof the second label are physically associated with the second nucleicacid target. For example, the first label can be part of a first labelprobe and the second label part of a second label probe, where the labelprobes are captured to the target nucleic acids.

In one class of embodiments, an average of at least four, eight, ortwelve copies of the first label are present in the cell per nucleotideof the first nucleic acid target over a region that spans at least 20contiguous nucleotides of the first nucleic acid target, and an averageof at least four, eight, or twelve copies of the second label arepresent in the cell per nucleotide of the second nucleic acid targetover a region that spans at least 20 contiguous nucleotides of thesecond nucleic acid target. In one embodiment, an average of at leastsixteen copies of the first label are present in the cell per nucleotideof the first nucleic acid target over a region that spans at least 20contiguous nucleotides of the first nucleic acid target, and an averageof at least sixteen copies of the second label are present in the cellper nucleotide of the second nucleic acid target over a region thatspans at least 20 contiguous nucleotides of the second nucleic acidtarget.

Essentially all of the features noted for the embodiments above apply tothese embodiments as well, as relevant, for example, with respect totype of labels, suspension of the cell, and/or the like. The regions ofthe first and second nucleic acid targets are typically regions coveredby a probe, primer, or similar polynucleotide employed to detect therespective target. The regions of the first and second nucleic acidtargets optionally span at least 25, 50, 100, 200, or more contiguousnucleotides and/or at most 2000, 1000, 500, 200, 100, 50, or fewernucleotides. A like density of labels is optionally captured to third,fourth, fifth, sixth, etc. nucleic acid targets. The compositionoptionally includes PCR primers, a thermostable polymerase, and/or thelike, in embodiments in which the targets are detected by multiplex insitu PCR.

Another aspect of the invention provides kits useful for practicing themethods. One general class of embodiments provides a kit for detecting afirst nucleic acid target and a second nucleic acid target in anindividual cell. The kit includes at least one reagent for fixing and/orpermeabilizing the cell, at least a first capture probe capable ofhybridizing to the first nucleic acid target, at least a second captureprobe capable of hybridizing to the second nucleic acid target, a firstlabel probe comprising a first label, and a second label probecomprising a second label, wherein a first signal from the first labelis distinguishable from a second signal from the second label, packagedin one or more containers.

Essentially all of the features noted for the embodiments above apply tothese embodiments as well, as relevant; for example, with respect tonumber of nucleic acid targets, configuration and number of the labeland capture probes, inclusion of preamplifiers and/or amplifiers,inclusion of blocking probes, inclusion of amplification reagents, typeof nucleic acid target, location of various targets on a single moleculeor on different molecules, type of labels, inclusion of optionalblocking probes, and/or the like. The kit optionally also includesinstructions for detecting the nucleic acid targets in the cell and/oridentifying the cell as being of a specified type, one or more bufferedsolutions (e.g., diluent, hybridization buffer, and/or wash buffer),reference cell(s) comprising one or more of the nucleic acid targets,and/or the like.

Another general class of embodiments provides a kit for detecting anindividual cell of a specified type from a mixture of cell types bydetecting a first nucleic acid target and a second nucleic acid target.The kit includes at least one reagent for fixing and/or permeabilizingthe cell, a first label probe comprising a first label (for detection ofthe first nucleic acid target), and a second label probe comprising asecond label (for detection of the second nucleic acid target), whereina first signal from the first label is distinguishable from a secondsignal from the second label, packaged in one or more containers. Thespecified type of cell is distinguishable from the other cell type(s) inthe mixture by presence, absence, or amount of the first nucleic acidtarget in the cell or by presence, absence, or amount of the secondnucleic acid target in the cell (that is, the two targets are redundantmarkers for the specified cell type).

Essentially all of the features noted for the embodiments above apply tothese embodiments as well, as relevant; for example, with respect tonumber of nucleic acid targets, inclusion of capture probes,configuration and number of the label and/or capture probes, inclusionof preamplifiers and/or amplifiers, inclusion of blocking probes,inclusion of amplification reagents, type of nucleic acid target,location of various targets on a single molecule or on differentmolecules, type of labels, inclusion of optional blocking probes, and/orthe like. The kit optionally also includes instructions for identifyingthe cell as being of the specified type, one or more buffered solutions(e.g., diluent, hybridization buffer, and/or wash buffer), referencecell(s) comprising one or more of the nucleic acid targets, and/or thelike.

IMPLEMENTATION, APPLICATIONS, AND ADVANTAGES

Various aspects of the invention are described in additional detailbelow. Exemplary embodiments and applications are also described.

The new technology (methods, compositions, systems, and kits), QMAGEX(Quantitative Multiplex Analysis of Gene Expression in Single Cell),disclosed herein is capable of detection and quantification of multiplenucleic acids within individual cells. The technology is significantlydifferent from existing ISH technology in several aspects, although theyboth can measure mRNA expression in individual cells. First, cellspreferably remain in suspension status during all or at least most ofthe assay steps in the assays of the present invention, which greatlyimproves assay hybridization kinetics, resulting in betterreproducibility and shorter assay time. Second, the instant technologyhas the capability for analyzing the expression of multiple mRNAtranscripts within cells simultaneously and quantitatively. This ishighly desirable, since, for example, detection of multiple tumor markergenes could greatly improve the accuracy of CTC identification (Mocellinet al., 2004) and greatly reduce the false positive rate. Quantitativeanalysis of gene expression level could not only further aid indiscriminating the CTC from other types of cells but also could help indistinguishing the type and source of primary tumors as well as thestages of tumor progression. Third, the instant technology enables theuse of a flow cytometer as the base for detection, which, compared withmicroscope-based detection instruments, offers higher throughput. Inaddition, the flow cytometer is capable of sorting out cells, e.g.,tumor cells, for further study. Subsequent to the detection andquantification of mRNA expression, isolation of the CTC or other cellsmay be advantageous for further identity confirmation or for additionalcytological and molecular analysis. Fourth, the instant technology hasvastly improved detection sensitivity and reproducibility, and iscapable of single copy gene detection and quantification. In addition,the instant technology uses a standard, generic set of probe labelingand detection technology (e.g., the same set of preamplifiers,amplifiers, and label probes can be used to detect multiple differentsets of nucleic acid targets, requiring only synthesis of a new set ofcapture probes for each new set of nucleic acid targets), and optionallyuses standardized procedures for cell fixation and permeation and forhybridization and washing. Furthermore, the technology can includebuilt-in internal controls for assay specificity and efficiency.

The instant technology can be used not only for the detection andenumeration of rare CTC in blood samples or other body fluids, but alsofor any type of rare cell identification and enumeration events.Applications include, but are not limited to: detection of minimalresidual disease in leukemia and lymphoma; recurrence monitoring afterchemotherapy treatment (Hess et al.); detection of other pre-cancerouscells, such as the detection of HPV-containing cervical cells in bodyfluids; detection of viral or bacterial nucleic acid in an infectedcell; detection of fetal cells in maternal blood; detection ofmicro-tumor lesions during early stage of tumor growth; or detection ofresidual tumor cells after surgery for margin management. In all ofthese cases, target cell specific gene expression is likely to be buriedin the background of large numbers of heterogeneous cell populations. Asa result, microarray or RT-PCR based expression analysis, which requirethe isolation of mRNA from a large population of cells, will havedifficulty detecting the presence of those rare cell events accuratelyor reliably, whereas the invented technology can readily be applied.

It should also be noted that although single cell detection andquantification of multiple mRNA transcripts is illustrated here as themain application, such technology is equally applicable to detection ofother rare cell events that include changes in chromosomal DNA orcellular nucleic acid content. Examples include, but are not limited to,detection of her-2/neu gene amplification, detection of Rb genedeletion, detection of somatic mutations, detection of chromosometranslation such as in chronic myelogenous leukemia (BCR-ABL), ordetection of HPV insertion to chromosomal DNA of cervical cancer cells.

Finally, the probe design, multiplexing and amplification aspects of theinstant technology can be applied in quantitative, multiplex geneexpression analysis and in measuring chromosomal DNA changes at a singlecell level in solid tissue sections, such as formalin-fixed, paraffinembedded (FFPE) tissue samples.

The QMAGEX technology comprises an assay and optional associatedapparatus to implement the assay in an automated fashion. FIG. 1illustrates major elements of the QMAGEX assay work flow, which, for oneexemplary embodiment in which amplifiers are employed, include:

Fixation and Permeation: Cells in the sample are fixed and permeated(permeabilized) in suspension. The fixation step immobilizes nucleicacids (e.g., mRNA or chromosomal DNA) and cross-links them to thecellular structure. Then the cell membrane is permeabilized so thattarget-specific nucleic acid probes and signal-generating particles,such as fluorescently labeled nucleic acid probes, can enter the celland bind to the target.

Denaturation: If the detection target is double-stranded chromosomalDNA, a denaturation step is added to convert the double-stranded targetinto single-stranded DNA, ready to be bound with the target-specificprobes.

Capture Probe Hybridization: Carefully selected target-specific captureprobes or probe sets are hybridized to the target nucleic acids. Thecapture probes serve to link the target molecules specifically tosignal-generating particles. The technology enables multiple targetgenes in the cell to be recognized by different probe setssimultaneously and with a high degree of specificity.

Signal Amplification: Signals from target molecules are amplified bybinding a large scaffold molecule, an amplifier, to the capture probesor probe sets. Each scaffold has multiple locations to accept labelprobes and signal-generating particles. In a multiplex assay, multipledistinct amplifiers are used.

Labeling: Label probes, to which signal generating particles (labels)are attached, hybridize to the amplifier in this step. In a multiplexassay, multiple distinct label probes are used.

Washing: The excess probes or signal generating particles that are notbound or that are nonspecifically bound to the cells are removed througha washing step, which reduces background noise and improves thedetection signal to noise ratio. Additional washing steps may be addedduring the capture probe hybridization or signal amplification steps tofurther enhance the assay performance.

Detection: The labeled suspension cells are detected using FluorescentActivated Cell Sorting (FACS) or a flow cytometer, or are immobilized ona solid surface and detected using a microscope or scanner basedinstrument.

In the following section, major elements of the QMAGEX technology willbe described in detail. In the following, the term label probe refers toan entity that binds to the target molecule, directly or indirectly, andenables the target to be detected by a readout instrument. The labelprobe, in general, comprises a nucleic acid or modified nucleic acidmolecule that binds to the target, directly or indirectly, and one ormore “signal generating particle” (i.e., label) that produces the signalrecognizable by the readout instrument. In indirect mode, the labelprobe can either be attached to the target molecule through binding to acapture probe directly or through binding to an amplifier that is inturn linked to a capture probe. Exemplary signal-generating particles(labels) include, but are not limited to, fluorescent molecules,nano-particles, radioactive isotopes, chemiluminescent molecules (e.g.,digoxigenin, dinitrophenyl). Fluorescent molecules include, but are notlimited to, fluorescein (FITC), cy3, cy5, alexa dyes, phycoerythrin,etc. Nano-particles include, but are not limited to, fluorescent quantumdots, scattering panicles, etc. The term capture probe refers to anucleic acid or a modified nucleic acid that links the target to aspecific type of label probe, directly or indirectly. The term “captureprobe set” refers to multiple nucleic acids or modified nucleic acidsthat link a target to a specific type of label probe, directly orindirectly, for increased assay sensitivity. The term amplifier refersto a large scaffold molecule(s) that binds to one or more capture probesor to a preamplifier on one side and to multiple label probes on anotherside.

Fixation

In this step, the nucleic acids are immobilized within cells bycross-linking them within the cellular structure. There are a variety ofwell known methods to fix cells in suspension with a fixative reagentand to block the endogenous RNase activities, which can be adapted foruse in the present invention. Fixative reagents include formalin(formaldehyde), paraformaldehyde, glutaraldehyde, ethanol, methanol,etc. One common fixative solution for tissue sections includes 0.25%glutaraldehyde and 4% paraformaldehyde in phosphate buffer. Anothercommon fixative solution for tissue sections includes 50% ethanol, 10%formalin (containing 37% formaldehyde), and 5% acetic acid. Differentcombinations of the fixative reagents at various concentrations areoptionally tested to find the optimal composition for fixing cells insuspension, using techniques well known in the art. Duration of thefixing treatment can also be optimized. A number of different RNaseinhibitors can be included in the fixative solution, such as RNAlater(Ambion), citric acid or LiCl, etc.

Permeation

Fixation results in cross-linking of the target nucleic acids withproteins or other cellular components within cells, which may hinder orprevent infiltration of the capture probes into the cells and mask thetarget molecules for hybridization. The assays of the invention thustypically include a follow-on permeation step to enable in-cellhybridization. One technique involves the application of heat forvarying lengths of time to break the cross-linking. This has beendemonstrated to increase the accessibility of the mRNA in the cells forhybridization. Detergents (e.g., Triton X-100 or SDS) and Proteinase Kcan also be used to increase the permeability of the fixed cells.Detergent treatment, usually with Triton X-100 or SDS, is frequentlyused to permeate the membranes by extracting the lipids. Proteinase K isa nonspecific protease that is active over a wide pH range and is noteasily inactivated. It is used to digest proteins that surround thetarget mRNA. Again, optimal concentrations and duration of treatment canbe experimentally determined as is well known in the art. A cell washingstep can follow, to remove the dissolved materials produced in thepermeation step.

Optionally, prior to fixation and permeation, cells in suspension arecollected and treated to inactivate RNase and/or to reduceautofluorescence. DEPC treatment (e.g. Braissant and Wahli (1988) “Asimplified in situ hybridization protocol using non-radioactivelylabeled probes to detect abundant and rare mRNAs on tissue sections”Biochemica 1:10-16) and RNAlater (Ambion, Inc.) have been demonstratedto be effective in stabilizing and protecting cellular RNA. Sodiumborohydride and high heat have also been shown to preserve the integrityof RNA and to reduce autofluorescence, facilitating the detection ofgenes expressed at a low level (Capodieci et al. (2005) “Gene expressionprofiling in single cells within tissue” Nat Methods 2(9):663-5). Othermethods of reducing cellular autofluorescence such as trypan blue(Mosiman et al. (1997) “Reducing cellular autofluorescence in flowcytometry: an in situ method” Cytometry 30(3):151-6) or singly labeledquencher oligonucleotide probe (Nolan et al. (2003) “A simple quenchingmethod for fluorescence background reduction and its application to thedirect, quantitative detection of specific mRNA” Anal Chem. 200375(22):6236-43) are optionally employed.

Capture Probe Hybridization

In this assay step, the capture probe or capture probe set binds to theintended target molecule by hybridization. One indicator for asuccessful target hybridization is specificity, i.e. the capture probesor probe sets should substantially only link the label probes to thespecific target molecule of interest, not to any other molecules. Probeselection and design are important in achieving specific hybridization.

Probe Selection and Design

The assays of the invention employ two types of approaches in probedesign to link the target nucleic acids in cells to signal generatingparticles: “direct labeling” and “indirect labeling”. In the directlabeling approach, the target molecule hybridizes to or captures one ormore label probes (LP) directly. The LPs contain the signal-generatingparticles (SGP), as shown in FIG. 2. A different LP needs to be used toattach additional SGP at different positions on the target molecule. Inorder to ensure hybridization specificity, the label probe is preferablystringently selected to ensure that it does not cross-hybridize withnonspecific nucleic acid sequences.

In the indirect labeling approach, an additional capture probe (CP) isemployed. An example is shown in FIG. 3. The target molecule capturesthe label probe through the capture probe. In each capture probe, thereis at least one section, T, complementary to a section on the targetmolecule, and another section, L, complementary to a section on thelabel probe. The T and L sections are connected by a section C. Toattach mote SGPs to different positions on the same target molecule,different capture probes are needed, but the label probe can remain thesame. The sequence of L is carefully selected to ensure that it does notcross-hybridize substantially with any sequences in the nucleic acids incells. In a further embodiment, the L portion of the capture probe andthe label probe contain chemically modified or nonnatural nucleotidesthat do not hybridize with natural nucleotides in cells. In anotherembodiment, L and the label probe (or a portion thereof) are not evennucleic acid sequences. For example, L can be a weak affinity bindingantibody that recognizes the signal generating probe, which in this caseis or includes an antigen; L can be covalently conjugated to anoligonucleotide that comprises the T section of the capture probe.Optionally, for two adjacent capture probes, the T sections hybridize tothe target and two of the low affinity binding antibody binds to theantigen on the label probe at the same time, which results in strongaffinity binding of the antigen. The capture and label probes arespecific for a target gene of interest. Multiple capture probes (probeset) can be bound to the same target gene of interest in order to attachmore signal generating particles for higher detection sensitivity. Inthis situation, the probe set for the same target gene can share thesame label probe.

Although both approaches can be used in the instant technology, theindirect capture approach is preferred because it enables the labelprobe to be target independent and further disclosure will show that itcan offer better specificity and sensitivity.

In a further indirect capture embodiment shown in FIG. 4, two adjacentcapture probes are incorporated in a probe set targeting a gene ofinterest. T₁ and T₂ are designed to be complementary to two unique andadjacent sections on the target nucleic acid. L₁ and L₂, which can bedifferent or the same, are complementary to two adjacent sections on thelabel probe. Their binding sections, T, L or both, are designed so thatthe linkage between the label probe and the target is unstable and tendsto fall off at hybridization temperature when only one of the captureprobes is in place. Such a design should enable exceptional specificitybecause the signal-generating label probe can only be attached to thetarget gene of interest when two independent capture probes bothrecognize the target and bind to the adjacent sequences or in very closeproximity of the target gene. In a further embodiment, the meltingtemperature, T_(m), of the T sections of the two capture probes aredesigned to be significantly above the hybridization temperature whilethe T_(m) of the L sections is below the hybridization temperature. As aresult, T sections bind to the target molecule strongly and stablyduring hybridization, while L sections bind to the label probe weaklyand unstably if only one of the capture probes is present. However, ifboth capture probes are present, the combination of L₁ and L₂ holds thelabel probe strongly and stably during hybridization. In anotherembodiment, T_(m) of the T sections is below hybridization temperaturewhile T_(m) of the L sections is substantially above. In the same way,the linkage between the label probe and the target can only survive thehybridization when both capture probes are hybridized to the target in acooperative fashion.

In another embodiment, three or more of the target nucleic acidspecific, neighboring capture probes are used for the stable capture ofone label probe within cells (FIG. 5). The basic design of the probes isthe same as discussed above, but the capture of one signal-generatingprobe should have even higher specificity than when two neighboringprobes are used since now three independent probes have to bind to thesame target molecule of interest in neighboring positions in order togenerate signal.

Multiplexing

To perform multiplexed detection for more than one target gene, e.g., asshown in FIG. 6, each target gene has to be specifically bound bydifferent capture and label probes. In addition, the signal generatingparticle (the label) attached to the label probe should providedistinctively different signals for each target that can be read by thedetection instrument. In the direct labeling approach (e.g., FIG. 6A),suitable label probes with minimal cross-hybridization can be harder tofind because each label probe has to be able to bind to the targetstrongly but not cross-hybridize to any other nucleic acid molecules inthe system. For this approach to provide optimal results, the targetbinding portion of the label probe should be judiciously designed sothat it does not substantially cross-hybridize with nonspecificsequences. In the indirect labeling approach (e.g., FIG. 6B), because ofthe unique multiple capture probe design approach, even when one captureprobe binds to a nonspecific target, it will not result in the bindingof the label probe to the nonspecific target. The assay specificity canbe greatly improved. Thus the capture probe design illustrated in FIG. 4and FIG. 5 is typically preferred in some multiplex assay applications.In one class of embodiments, the signal-generating particles attached todifferent target genes are different fluorescent molecules withdistinctive emission spectra.

The capacity of the instant technology to measure more than oneparameter simultaneously can enable detection of rare cells in a largeheterogeneous cell population. As noted above, the concentration of CTCis estimated to be in the range of one tumor cell among every 10⁶-10⁷normal blood cells. In existing FACS based immunoassays, on the otherhand, random dye aggregation in cells may produce one false positivecell count in every ten thousand cells. Such an assay can thus not beused for CTC detection due to the unacceptably high false positiverates. This problem can be solved elegantly using the instanttechnology. In one particular embodiment, expression of more than onetumor genes are used as the targets for multiplex detection. Only cellsthat express all the target genes are counted as tumor cells. In thisway, the false positive rate of the CTC detection can be dramaticallyreduced. For example, since dye aggregation in cells is a random event,if the false positive rate of a single color detection is 10⁴, the falsepositive rate for two color or three color detection can be as low as10⁻⁸ or 10⁻¹², respectively. In situations where the relative levels ofexpression of the target genes are known, these relative levels can bemeasured using the multiplex detection methods disclosed herein and theinformation can be used to further reduce the false positive rate of thedetection.

In another embodiment, schematically illustrated in FIG. 7A, more thanone signal-generating particles are linked to the same target nucleicacid. These panicles generate distinct signals in the detectioninstrument. The relative strengths of these signals can bepre-determined by designing the number of each type of particlesattached to the target. The number of signal-generating particles on atarget can be controlled in probe design by changing the number of probesets or employing different signal amplification methods, e.g., asdescribed in the following section. The rare cells are identified onlywhen the relative signal strengths of these particles measured by thedetection instrument equal the pre-determined values. This embodiment isuseful when there are not enough suitable markers or when theirexpression levels are unknown in a particular type of rare cells. In yetanother embodiment, shown in FIG. 7B, the same set of signal-generatingparticles are attached to more than one target. The relative signalstrengths of the particle set are controlled to be the same on allselected targets. This embodiment is useful in situations in which therare cell is identified when any of the target molecules are present. Inyet another embodiment, depicted in FIG. 7C, each target molecule has aset of signal generating particles attached to it, but the particle setsare distinctively different from target to target.

The detection of multiple target nucleic acid species of interest can beapplied to quantitative measurement of one target. Due to differentsample and experimental conditions, the abundance of a particular targetmolecule in a cell normally cannot be determined precisely through thedetection of the signal level associated with the target. However, moreprecise measurement can be accomplished by normalizing the signal of agene of interest to that of a reference/housekeeping gene. Areference/housekeeping gene is defined as a gene that is generallyalways present or expressed in cells. The expression of thereference/housekeeping gene is generally constitutive and tends not tochange under different biological conditions. 18S, 28S, GAPD, ACTB, PPIBetc. have generally been considered as reference or housekeeping genes,and they have been used in normalizing gene expression data generatedfrom different samples and/or under varying assay conditions.

In another embodiment, a special label probe set can be designed thatdoes not bind to any capture probe or target specifically. The signalassociated to this label probe can be used to establish the backgroundof hybridization signal in individual cells. Thus the abundance of aparticular target molecule can be quantitatively determined by firstsubtracting the background hybridization signal, then normalizingagainst the background subtracted reference/housekeeping genehybridization signal.

In yet another embodiment, two or more chromosomal DNA sequences ofinterest can be detected simultaneously in cells. In the detection ofmultiple DNA sequences in cells, the label probes for the DNA sequencesare distinct from each other and they do not cross-hybridize with eachother. In embodiments in which cooperative indirect capture is employed,because of the design scheme, even when one probe binds to a nonspecificDNA sequence, it will not result in the capture of the signal-generatingprobe to the nonspecific DNA sequences.

In yet another embodiment, the detection of multiple target chromosomalDNA sequences of interest enables quantitative analysis of geneamplification, gene deletion, or gene translocations in single cells.This is accomplished by normalizing the signal of a gene of interest tothat of a reference gene. The signal ratio of the gene of interest tothe reference gene for a particular cell of interest is compared withthe ratio in reference cells. A reference gene is defined as a gene thatstably maintains its copy numbers in the genomic DNA. A reference cellis defined as a cell that contains the normal copy number of the gene ofinterest and the reference gene. If the signal ratio is higher in thecells of interest in comparison to the reference cells, geneamplification is detected. If the ratio is lower in the cells ofinterest in comparison to the reference cells, then gene deletion isdetected.

Signal Amplification & Labeling

The direct labeling approach depicted in FIG. 2 and FIG. 6A offers onlylimited sensitivity because only a relatively small number ofsignal-generating particles (labels) can be attached to each labelprobe. One way to increase sensitivity is to use in vitro transcribedRNA that incorporates signal-generating panicles, but specificity willsuffer as a result.

The “indirect labeling” approach not only can improve specificity asdescribed above but also can be used to improve the detectionsensitivity. In this approach, the label probe is hybridized orconnected to an amplifier molecule, which provides many more attachmentlocations for label probes. The structure and attachment method of theamplifier can take many forms. FIGS. 8A-8D show a number ofamplification schemes as illustrative examples. In FIG. 8A, multiplesingly-labeled label probes bind to the amplifier. In FIG. 8B, multiplemultiply-labeled label probes bind to the amplifier. In FIG. 8C,multiple singly-labeled label probes bind to the amplifier, and multiplecopies of the amplifier are bound to a preamplifier. In one particularembodiment, the amplifier is one or multiple branched DNA molecules(FIG. 8D). The sequence of the label probe is preferably selectedcarefully so that it does not substantially cross-hybridize with anyendogenous nucleic acids in the cell. In fact, the label probe does nothave to be a natural polynucleotide molecule. Chemical modification ofthe molecule, for example, inclusion of nonnatural nucleotides, canensure that the label probe only hybridizes to the amplifier and not tonucleic acid molecules naturally occurring in the cells. In multiplexassays, distinct amplifiers and label probes will be designed and usedfor the different targets.

In one embodiment, as schematically illustrated in FIGS. 9A-9C, acircular polynucleotide molecule is captured by the capture probe set.Along the circle, there can be one sequence or more than one repeat ofthe same sequence that binds to label probe (FIG. 9A). In the signalamplification step of the assay, a rolling circle amplificationprocedure (Larsson et al, 2004) is carried out. As the result of thisprocedure, a long chain polynucleotide molecule attached to the captureprobes is produced (FIG. 9B). There are many repeating sequences alongthe chain, on which label probes can be attached by hybridization (FIG.9C). In multiplex assays, distinct capture probes, rolling circles, andlabel probes will be designed and used.

In one embodiment, a portion of the signal-generating probe can bePCR-amplified. In another embodiment, each portion of multiplesignal-generating probes can be PCR-amplified simultaneously.

Although a specific capture approach (indirect labeling with captureprobe pairs) has been used to illustrate the labeling and amplificationschemes in FIGS. 8A-8D and FIGS. 9A-9C, it is important to note that anyother probe capture approaches, direct or indirect, described inprevious sections can be used in combination with the labeling andamplification schemes described in these sections. The capture probe,labeling methods, and amplifier configurations described above areindependent of each other and can be used in any combination in aparticular assay design.

Hybridization Conditions

The composition of the hybridization solution can affect efficiency ofthe hybridization process. Hybridization typically depends on theability of the oligonucleotide to anneal to a complementary mRNA strandbelow its melting point (T_(m)). The value of the T_(m) is thetemperature at which half of the oligonucleotide duplex is present in asingle stranded form. The factors that influence the hybridization ofthe oligonucleotide probes to the target nucleic acids can includetemperature, pH, monovalent cation concentration, presence of organicsolvents, etc. A typical hybridization solution can contain some or allof the following reagents, e.g., dextran sulfate, formamide, DTT(dithiothreitol), SSC (NaCl plus sodium citrate), EDTA, etc. Othercomponents can also be added to decrease the chance of nonspecificbinding of the oligonucleotide probes, including, e.g., single-strandedDMA, tRNA acting as a carrier RNA, polyA, Denhardt's solution, etc.Exemplary hybridization conditions can be found in the art and/ordetermined empirically as well known in the art. See, e.g., U.S. patentapplication publication 2002/0172950, Player et al. (2001) J. Histochem.Cytochem. 49:603-611, and Kenny et al. (2002) J. Histochem. Cytochem.50:1219-1227, which also describe fixation, permeabilization, andwashing.

An additional prehybridization is optionally carried out to reducebackground staining. Prehybridization involves incubating the fixedtissue or cells with a solution that is composed of all the elements ofthe hybridization solution, minus the probe.

Washing

Following the labeling step, the cells are preferably washed to removeunbound probes or probes which have loosely bound to imperfectly matchedsequences. Washing is generally started with a low stringency washbuffer such as 2×SSC+1 mM EDTA (1×SSC is 0.15M NaCl, 0.015M Na-citrate),then followed by washing with higher stringency wash buffer such as0.2×SSC+1 mM EDTA or 0.1×SSC+1 mM EDTA.

Washing is important in reducing background noise, improving signal tonoise ratio of and quantification with the assay. Established washingprocedures can be found, e.g., in Bauman and Bentvelzen (1988) “Flowcytometric detection of ribosomal RNA in suspended cells by fluorescentin situ hybridization” Cytometry 9(6):517-24 and Yu et al. (1992)“Sensitive detection of RNAs in single cells by flow cytometry” NucleicAcids Res. 20(1):83-8.

Washing can be accomplished by executing a suitable number of washingcycles, i.e., one or more. Each cycle in general includes the followingsteps: mixing the cells with a suitable buffer solution, detachingnon-specifically bound materials from the cells, and removing the buffertogether with the waste. Each step is described in more detail below.

Mix the cells with wash buffer: In some assays, the cells areimmobilized on the surface of a substrate before being washed. In suchcases, the washing buffer is mixed together with the substrate surface.In many other embodiments, the cells to be washed are free-floating. Thewashing buffer is added to cell pellets or to the solution in which thecells are floating.

Detach non-specifically bound materials from cells: Any of a number oftechniques can be employed here to reduce nonspecific binding after cellpermeability treatment and probe hybridization to encouragenon-specifically bound probes to detach from the cells and dissolve intothe wash buffer. These include raising the temperature to somewhere justbelow the melting temperature of the specifically bound probes andemploying agitation using a magnetic or mechanical stirrer orperturbation with sonic or ultrasonic waves. Agitation of the mixturecan also be achieved by shaking the container with a rocking or vortexmotion.

Remove buffer together with waste: Any convenient method can be employedto separate and remove the washing buffer and waste from the targetcells in the sample. For example, the floating cells or substrates thatthe cells bound to are separated from the buffer and waste throughcentrifugation. After the spin, the cells or substrates form a pellet atthe bottom of the container. The buffer and waste are decanted from thetop.

As another example, the mixture is optionally transferred to (or formedin) a container the bottom of which is made of a porous membrane. Thepore size of the membrane is chosen to be smaller than the target cellsor the substrates that the cells are bound to but large enough to allowfor debris and other waste materials to pass through. To remove thewaste, the air or liquid pressure is optionally adjusted such that thepressure is higher inside the container than outside, thus driving thebuffer and waste out of the container while the membrane retains thetarget cells inside. The waste can also be removed, e.g., by filteringthe buffer and waste through the membrane driven by the force of gravityor by centrifugal force.

As yet another example, the cells can be immobilized on the surface of alarge substrate, for example, a slide or the bottom of a container,through cell fixing or affinity attachment utilizing surface proteins.The buffer and waste can be removed directly by either using a vacuum todecant from the top or by turning the container upside down. As yetanother example, the cells are optionally immobilized on magnetic beads,e.g., by either chemical fixing or surface protein affinity attachment.The beads can then be immobilized on the container by attaching amagnetic field on the container. The buffer and waste can then beremoved directly without the loss of cells the same way as described inthe previous example. As yet another example, the nonspecifically boundprobes within cells are induced to migrate out of the cells byelectrophoretic methods while the specifically hound probes remain.

As stated before, a washing cycle is completed by conducting each of thethree steps above, and the washing procedure is accomplished byexecuting one or more (e.g., several) such washing cycles. Differentwashing buffers, detachment, or waste removal techniques may be used indifferent washing cycles.

Detection

In the instant technology, the target cells that have signal-generatingparticles (labels) specifically hybridized to nucleic acid targets inthem can be identified out of a large heterogeneous population afternon-specifically bound probes and other wastes are removed throughwashing. Essentially any convenient method for the detection andidentification can be employed.

In one embodiment, the suspension cells are immobilized onto a solidsubstrate after the labeling or washing step described above. Thedetection can be achieved using microscope based instruments.Specifically, in cases where the signal generated by the probes ischemiluminescent light, an imaging microscope with a CCD camera or ascanning microscope can be used to convert the light signal into digitalinformation. In cases where the probe carries a label emitting afluorescent signal, a fluorescent imaging or scanning microscope basedinstrument can be used for detection. In addition, since the targetcells are, in general, rare among a large cell population, automaticevent finding algorithms can be used to automatically identify and countthe number of target cells in the population. Cells in suspension can beimmobilized onto solid surfaces by any of a number of techniques. In oneembodiment, a container with large flat bottom surface is used to holdthe solution with the suspended cells. The container is then centrifugedto force the floating cells to settle on the bottom. If the surface issufficiently large in comparison to the concentration of cells in thesolution, cells are not likely to overlap on the bottom surface. In mostcases, even if the cells overlap, the target cells will not because theyare relatively rare in a large population. In another embodiment,suspended cells are cytospun onto a flat surface. After removal offluids, the cells are immobilized on the surface by surface tension.

In preferred embodiments of this invention, cells are floating (insuspension) or are immobilized on floating substrates, such as beads, sothat pre-detection procedures, such as hybridization and washing, can becarried out efficiently in solution. There are several methods to detectrare target cells out of a large floating cell population. The preferredmethod is to use a detection system based on the concept of flowcytometry, where the floating cells or substrates are streamlined andpass in front of excitation and detection optics one by one. The targetcells are identified through the optical signal emitted by the probesspecifically bound to the nucleic acid targets in the cells. The opticalsignal can, e.g., be luminescent light or fluorescent light of aspecific wavelength.

Advantages

In summary, the instant QMAGEX technology has a number of uniqueelements that enable multiplex nucleic acid detection in single cellsand detection of target cells. These elements include the following.

Nucleic acid molecules immobilized inside cells are used as markers forthe identification of CTC (or other cell types). Compared with proteinbased markers, nucleic acids are more stable, widely available, andprovide better signal to noise ratio in detection. In addition, thedetection technique can be readily applied to a wide range of tumors oreven other applications related to cell identification orclassification. As another advantage, nucleic acid molecules arequantifiably measured at an individual cell level, instead of in a mixedcell population. This feature ensures that the cell as a key functionalunit in the biological system is preserved for study. In manyapplications involving a mixed population of cells, this feature can bevery useful in extracting real, useful information out of the assay.(For example, a CTC can be identified based on detection of the presenceor expression level(s) of a set of nucleic acid marker(s) in the cell;the presence or copy number of additional nucleic acids in the cell canthen provide additional information useful in diagnosis, predictingoutcome, or the like.)

Cells optionally remain in suspension or in pellets that can bere-suspended in all steps of the assay before final detection. Thisfeature significantly improves assay kinetics, simplifies the process,enhances the reproducibility, and keeps the cell in its most functionalrelevant status. On the other hand, significant aspects of theinvention, including probe selection and design, multiplexing,amplification and labeling, can be applied directly to in situhybridization technique for the detection and enumeration of rare cellsin tissue samples.

A unique indirect capture probe design approach is optionally employedto achieve exceptional target hybridization specificity, which resultsin better signal to noise ratio in detection.

The assays enable the detection of multiple target genes or multipleparameters on the same gene simultaneously. This feature benefits thedetection of rare cells such as CTC in a number of ways. First, it canreduce the false positive rate, which is essential in cancerdiagnostics. Second, it can provide additional, clinically importantinformation related to the detected tumor cell, which may include theprogression stage and/or original type and source of the primary tumor.

The invented technology incorporates a signal amplification scheme,which boosts the detection sensitivity and enables the detection of rarecells among a large number of normal cells with high confidence.

Detection can be implemented on FACS or flow cytometer based instrumentsor on microscope based platforms. The former can be fully automated andprovides fast detection and the additional benefit of sorting outidentified cells for further study, if desired. The latter platform ismore widely available and has the benefit of allowing final manualidentification through morphology.

Systems

In one aspect, the invention provides systems and apparatus configuredto carry out the procedures of the novel assays. The apparatus or systemcomprises one or more (and preferably all) of at least the followingelements.

Fluid handling: The apparatus optionally includes a subsystem that canadd reagents, and if required by the assay, decant fluids from thesample container (e.g., a removable or fixed, disposable or reusablecontainer, for example a sample tube, multiwell plate, or the like). Thesubsystem can be based on a pipette style fluid transfer system wheredifferent fluids are handled by one pump head with disposable tips. Asan alternative example, each reagent may have its own dedicated fluidchannel.

Mixing and agitation: The apparatus optionally includes a device to mixdifferent reagents in the sample solution and encourage anynon-specifically bound material to detach from the cells. The device mayhave a mechanism to introduce a vortex or rocking motion to the holderof the sample container or to couple sound or ultrasound to thecontainer. Alternatively, a magnetic stirrer can be put into the samplecontainer and be driven by rotating magnetic field produced by anelement installed in a holder for the container.

Temperature control: The temperature of the sample can be controlled toa level above the room temperature by installing a heater and atemperature probe to the chamber that holds the sample container. Apeltier device can be used to control the temperature to a level aboveor below ambient. Temperature control is important, e.g., forperformance of the hybridization and washing procedures in the assays.

Cell and waste fluid separation: The apparatus optionally includes adevice that can remove waste fluid from the sample mixture whileretaining cells for further analysis. The device may comprise a samplecontainer that has a porous membrane as its bottom. The pore size of themembrane is smaller than the cells but larger than the waste material inthe mixed solution. The space below the membrane can be sealed andconnected to a vacuum pump. As an alternative example, the space abovethe membrane can be sealed and connected to a positive pressure source.In a different embodiment, the device can comprise a centrifuge. Thecontainer with the membrane bottom is loaded into the centrifuge, whichspins to force the waste solution to filter out through the membrane. Inanother configuration of this device, the sample container has a solidbottom. Cells deposit at the bottom after centrifugation, and the wastesolution is decanted from the top by the fluid handling subsystemdescribed above.

This device can also perform a function that prepares the sample forfinal readout. In embodiments where the readout is by microscopy, thecells are typically deposited and attached to a flat surface. Acentrifuge in the device can achieve this if the bottom of the containeris flat. In another approach, a flat plate can spin within its plane,and the system can employ the fluid handling device to drop the solutioncontaining the cells at the center of the spin. The cells will be evenlyspun on the plate surface.

Detection: The detection element of the invented apparatus can beintegrated with the rest of the system, or alternatively it can beseparate from the rest of the subsystems described above. In oneembodiment, the readout device is based on a microscope, which may be animaging or scanning microscope. In another embodiment, the device isbased on a fluorescent imaging or scanning microscope with multipleexcitation and readout wavelengths for different probes. In a preferredembodiment, the readout device is based on flow cytometry. The cytometryapproach is preferred because it can read floating cells directly out offluid at multiple wavelengths thus greatly improving the efficiency ofthe assay.

All of the above elements can be integrated into one instrument.Alternatively, these elements may be included in a number ofinstruments, which work together as a system to perform the assay. FIG.10 illustrates one particular exemplary embodiment of the instrumentconfiguration. In this particular configuration, the sample is held in acontainer (sample test tube) with a membrane bottom. Reagents are addedfrom the top of the tube using a pump through a multiport valve. Wasteis removed from bottom by vacuum. The holder for the sample container isfixed on an agitation table and the space around the sample istemperature controlled (temp controlled zone) by the temperaturecontroller. The fluid handling element can introduce reagents (fixationand permeation reagents, hybridization buffer, probes sets, and washbuffer) into the sample tube, remove waste into a waste container, andfeed cells to a flow cytometer for detection.

One class of embodiments provides a system comprising a holderconfigured to accept a sample container; a temperature controllerconfigured to maintain the sample container at a selected temperature(e.g., a temperature selected by a user of the system or a presettemperature, different temperatures are optionally selected fordifferent steps in an assay procedure); a fluid handling element fluidlyconnected to the sample container and configured to add fluid to and/orremove fluid from the sample container; a mixing element configured tomix (e.g., stir or agitate) contents of the sample container; and adetector for detecting one or more signals from within individual cells,wherein the detector is optionally fluidly connected to the samplecontainer. One of more fluid reservoirs (e.g., for fixation orpermeabilization reagents, wash buffer, probe sets, and/or waste) areoptionally fluidly connected to the sample container.

A system of the invention optionally includes a computer. The computercan include appropriate software for receiving user instructions, eitherin the form of user input into a set of parameter fields, e.g., in aGUI, or in the form of preprogrammed instructions, e.g., preprogrammedfor a variety of different specific operations. The software optionallyconverts these instructions to appropriate language for controlling theoperation of components of the system (e.g., for controlling a fluidhandling element and/or laser). The computer can also receive data fromother components of the system, e.g., from a detector, and can interpretthe data, provide it to a user in a human readable format, or use thatdata to initiate further operations, in accordance with any programmingby the user.

Nucleic Acid Targets

As noted, a nucleic acid target can be essentially any nucleic acid thatis desirably detected in a cell. Choice of targets will obviously dependon the desired application, e.g., expression analysis, diseasediagnosis, staging, or prognosis, target identification or validation,pathway analysis, drug screening, drug efficacy studies, or any of manyother applications. Large numbers of suitable targets have beendescribed in the art, and many more can be identified using standardtechniques.

For detection of CTC, as just one example, a variety of suitable nucleicacid targets are known. For example, a multiplex panel of markers forCTC detection could include one or more of the following markers:epithelial cell-specific (e.g. CK19, Muc1, EpCAM), blood cell-specificas negative selection (e.g. CD45), tumor origin-specific (e.g. PSA,PSMA, HPN for prostate cancer and mam, mamB, her-2 for breast cancer),proliferating potential-specific (e.g. Ki-67, CEA, CA15-3), apoptosismarkers (e.g. BCL-2, BCL-XL), and other markers for metastatic, geneticand epigenetic changes. As another example, targets can include HOXB13and IL17BR mRNAs, whose ratio in primary tumor has been shown to predictclinical outcome of breast cancer patients treated with tamoxifen (Ma etal. (2004) “A two-gene expression ratio predicts clinical outcome inbreast cancer patients treated with tamoxifen” Cancer Cell 5(6):607-16and Goetz et al. (2006) “A Two-Gene Expression Ratio of Homeobox 13 andInterleukin-17B Receptor for Prediction of Recurrence and Survival inWomen Receiving Adjuvant Tamoxifen” Clin Cancer Res 12:2080-2087). Seealso, e.g., Gewanter, R. M., A. E. Katz, et al. (2003) “RT-PCR for PSAas a prognostic factor for patients with clinically localized prostatecancer treated with radiotherapy” Urology 61(5):967-71; Giatromanolakiet al. (2004) “Assessment of highly angiogenic and disseminated in theperipheral blood disease in breast cancer patients predicts forresistance to adjuvant chemotherapy and early relapse” Int J Cancer108(4):620-7; Halabi et al. (2003) “Prognostic significance of reversetranscriptase polymerase chain reaction for prostate-specific antigen inmetastatic prostate cancer: a nested study within CALGB 9583” J ClinOncol 21(3):490-5; Hardingham et al. (2000) “Molecular detection ofblood-borne epithelial cells in colorectal cancer patients and inpatients with benign bowel disease” Int J Cancer 89(1):8-13; Hayes etal. (2002) “Monitoring expression of HER-2 on circulating epithelialcells in patients with advanced breast cancer” Int J Oncol 21(5):1111-7; Jotsuka, et al. (2004) “Persistent evidence of circulating tumorcells detected by means of RT-PCR for CEA mRNA predicts early relapse: aprospective study in node-negative breast cancer” Surgery 135(4):419-26;Allen-Mersh T et al. (2003) “Colorectal cancer recurrence is predictedby RT-PCR detection of circulating cancer cells at 24 hours afterprimary excision” ASCO meeting, Chicago, May 2003; Shariat et al. (2003)“Early postoperative peripheral blood reverse transcription PCR assayfor prostate-specific antigen is associated with prostate cancerprogression in patients undergoing radical prostatectomy” Cancer Res63(18):5874-8; Smith et al. (2000) “Response of circulating tumor cellsto systemic therapy in patients with metastatic breast cancer:comparison of quantitative polymerase chain reaction andimmunocytochemical techniques” J Clin Oncol 18(7):1432-9; Stathopoulouet al. (2002) “Molecular detection of cytokeratin-19-positive cells inthe peripheral blood of patients with operable breast cancer: evaluationof their prognostic significance” J Clin Oncol 20(16):3404-12; andXenidis et al. (2003) “Peripheral blood circulating cytokeratin-19mRNA-positive cells after the completion of adjuvant chemotherapy inpatients with operable breast cancer” Ann Oncol 14(6):849-55.

One preferred class of nucleic acid targets to be detected in themethods herein are those involved in cancer. Any nucleic acid that isassociated with cancer can be detected in the methods of the invention,e.g., those that encode over expressed or mutated polypeptide growthfactors (e.g., sis), overexpressed or mutated growth factor receptors(e.g., erb-B1), over expressed or mutated signal transduction proteinssuch as G-proteins (e.g., Ras), or non-receptor tyrosine kinases (e.g.,abl), or over expressed or mutated regulatory proteins (e.g., myc, myb,jun, fos, etc.) and/or the like. In general, cancer can often be linkedto signal transduction molecules and corresponding oncogene products,e.g., nucleic acids encoding Mos, Ras, Raf, and Met; and transcriptionalactivators and suppressors, e.g., p53, Tat, Fos, Myc, Jun, Myb, Rel,and/or nuclear receptors. p53, colloquially referred to as the“molecular policeman” of the cell, is of particular relevance, as about50% of all known cancers can be traced to one or more genetic lesion inp53.

Taking one class of genes that are relevant to cancer as an example fordiscussion, many nuclear hormone receptors have been described in detailand the mechanisms by which these receptors can be modified to conferoncogenic activity have been worked out. For example, the physiologicaland molecular basis of thyroid hormone action is reviewed in Yen (2001)“Physiological and Molecular Basis of Thyroid Hormone Action”Physiological Reviews 81(3): 1097-1142, and the references citedtherein. Known and well characterized nuclear receptors include thosefor glucocorticoids (GRs), androgens (ARs), mineralocorticoids (MRs),progestins (PRs), estrogens (ERs), thyroid hormones (TRs), vitamin D(VDRs), retinoids (RARs and RXRs), and the peroxisome proliferatoractivated receptors (PPARs) that bind eicosanoids. The so called “orphannuclear receptors” are also part of the nuclear receptor superfamily,and are structurally homologous to classic nuclear receptors, such assteroid and thyroid receptors. Nucleic acids that encode any of thesereceptors, or oncogenic forms thereof, can be detected in the methods ofthe invention. About 40% of all pharmaceutical treatments currentlyavailable are agonists or antagonists of nuclear receptors and/oroncogenic forms thereof, underscoring the relative importance of thesereceptors (and their coding nucleic acids) as targets for analysis bythe methods of the invention.

One exemplary class of target nucleic acids are those that arediagnostic of colon cancer, e.g., in samples derived from stool. Coloncancer is a common disease that can be sporadic or inherited. Themolecular basis of various patterns of colon cancer is known in somedetail. In general, germline mutations are the basis of inherited coloncancer syndromes, while an accumulation of somatic mutations is thebasis of sporadic colon cancer. In Ashkenazi Jews, a mutation that waspreviously thought to be a polymorphism may cause familial colon cancer.Mutations of at least three different classes of genes have beendescribed in colon cancer etiology: oncogenes, suppressor genes, andmismatch repair genes. One example nucleic acid encodes DCC (deleted incolon cancer), a cell adhesion molecule with homology to fibronectin. Anadditional form of colon cancer is an autosomal dominant gene, hMSH2,that comprises a lesion. Familial adenomatous polyposis is another formof colon cancer with a lesion in the MCC locus on chromosome number 5.For additional details on colon cancer, see, Calvert et al. (2002) “TheGenetics of Colorectal Cancer” Annals of Internal Medicine 137 (7):603-612 and the references cited therein. For a variety of colon cancersand colon cancer markers that can be detected in stool, see, e.g.,Boland (2002) “Advances in Colorectal Cancer Screening: Molecular Basisfor Stool-Based DNA Tests for Colorectal Cancer: A Primer forClinicians” Reviews In Gastroenterological Disorders Volume 2, Supp. 1and the references cited therein. As with other cancers, mutations in avariety of other genes that correlate with cancer, such as Ras and p53,are useful diagnostic indicators for cancer.

Cervical cancer is another exemplary target for detection, e.g., bydetection of nucleic acids that are diagnostic of such cancer in samplesobtained from vaginal secretions. Cervical cancer can be caused by thepapova virus (e.g., human papilloma virus) and has two oncogenes, E6 andE7. E6 binds to and removes p53 and E7 binds to and removes PRB. Theloss of p53 and uncontrolled action of E2F/DP growth factors without theregulation of pRB is one mechanism that leads to cervical cancer.

Another exemplary target for detection by the methods of the inventionis retinoblastoma, e.g., in samples derived from tears. Retinoblastomais a tumor of the eyes which results from inactivation of the pRB gene.It has been found to transmit heritably when a parent has a mutated pRBgene (and, of course, somatic mutation can cause non-heritable forms ofthe cancer).

Neurofibromatosis Type 1 can be detected in the methods of theinvention. The NF1 gene is inactivated, which activates the GTPaseactivity of the ras oncogene. If NF1 is missing, ras is overactive andcauses neural tumors. The methods of the invention can be used to detectNeurofibromatosis Type 1 in CSF or via tissue sampling.

Many other forms of cancer are known and can be found by detectingassociated genetic lesions using the methods of the invention. Cancersthat can be detected by detecting appropriate lesions include cancers ofthe lymph, blood, stomach, gut, colon, testicles, pancreas, bladder,cervix, uterus, skin, and essentially all others for which a knowngenetic lesion exists. For a review of the topic, see, e.g., TheMolecular Basis of Human Cancer Coleman and Tsongalis (Eds) HumanaPress; ISBN: 0896036340; 1st edition (August 2001).

Similarly, nucleic acids from pathogenic or infectious organisms can bedetected by the methods of the invention, e.g., for infectious fungi,e.g., Aspergillus, or Candida species; bacteria, particularly E. coli,which serves a model for pathogenic bacteria (and, of course certainstrains of which are pathogenic), as well as medically importantbacteria such as Staphylococci (e.g., aureus), or Streptococci (e.g.,pneumoniae); protozoa such as sporozoa (e.g., Plasmodia), rhizopods(e.g., Entamoeba) and flagellates (Trypanosoma, Leishmania, Trichomonas,Giardia, etc.); viruses such as (+) RNA viruses (examples includePoxviruses e.g., vaccinia; Picornaviruses, e.g. polio; Togaviruses,e.g., rubella; Flaviviruses, e.g., HCV; and Coronaviruses), (−) RNAviruses (e.g., Rhabdoviruses, e.g., VSV; Paroxymyxoviruses, e.g., RSV;Orthomyxoviruses, e.g., influenza; Bunyaviruses; and Arenaviruses),dsDNA viruses (Reoviruses, for example), RNA to DNA viruses, i.e.,Retroviruses, e.g., HIV and HTLV, and certain DNA to RNA viruses such asHepatitis B.

As noted previously, gene amplification or deletion events can bedetected at a chromosomal level using the methods of the invention, ascan altered or abnormal expression levels. One preferred class ofnucleic acid targets to be detected in the methods herein includeoncogenes or tumor suppressor genes subject to such amplification ordeletion. Exemplary nucleic acid targets include, but are not limitedto, integrin (e.g., deletion), receptor tyrosine kinases (RTKs; e.g.,amplification, point mutation, translocation, or increased expression),NF1 (e.g., deletion or point mutation), Akt (e.g., amplification, pointmutation, or increased expression), PTEN (e.g., deletion or pointmutation), MDM2 (e.g., amplification), SOX (e.g., amplification), RAR(e.g., amplification), CDK2 (e.g., amplification or increasedexpression), Cyclin D (e.g., amplification or translocation), Cyclin E(e.g., amplification), Aurora A (e.g., amplification or increasedexpression), P53 (e.g., deletion or point mutation), NBS1 (e.g.,deletion or point mutation), Gli (e.g., amplification or translocation),Myc (e.g., amplification or point mutation), HPV-E7 (e.g., viralinfection), and HPV-E6 (e.g., viral infection).

For embodiments in which a nucleic acid target is used as a reference,suitable reference nucleic acids have similarly been described in theart or can be determined. For example, a variety of genes whose copynumber is stably maintained in various tumor cells is known in the art.Housekeeping genes whose transcripts can serve as references in geneexpression analyses include, for example, 18S rRNA, 28S rRNA, GAPD,ACTB, and PPIB. Additional similar nucleic acids have been described inthe art and can be adapted to the practice of the present invention.

Labels

A wide variety of labels are well known in the art and can be adapted tothe practice of the present invention. For example, luminescent labelsand light-scattering labels (e.g., colloidal gold particles) have beendescribed. See, e.g., Csaki et al. (2002) “Gold nanoparticles as novellabel for DNA diagnostics” Expert Rev Mol Diagn 2:187-93.

As another example, a number of fluorescent labels are well known in theart, including but not limited to, hydrophobic fluorophores (e.g.,phycoerythrin, rhodamine, Alexa Fluor 488 and fluorescein), greenfluorescent protein (GFP) and variants thereof (e.g., cyan fluorescentprotein and yellow fluorescent protein), and quantum dots. See e.g., TheHandbook: A Guide to Fluorescent Probes and Labeling Technologies, TenthEdition or Web Edition (2006) from Invitrogen (available on the worldwide web at probes.invitrogen.com/handbook), for descriptions offluorophores emitting at various different wavelengths (including tandemconjugates of fluorophores that can facilitate simultaneous excitationand detection of multiple labeled species). For use of quantum dots Aslabels for biomolecules, see e.g., Dubertret et al. (2002) Science298:1759; Nature Biotechnology (2003) 21:41-46; and Nature Biotechnology(2003) 21:47-51.

Labels can be introduced to molecules, e.g. polynucleotides, duringsynthesis or by postsynthetic reactions by techniques established in theart. For example, kits for fluorescently labeling polynucleotides withvarious fluorophores are available from Molecular Probes, Inc. ((www.)molecularprobes.com), and fluorophore-containing phosphoramidites foruse in nucleic acid synthesis are commercially available. Similarly,signals from the labels (e.g., absorption by and/or fluorescent emissionfrom a fluorescent label) can be detected by essentially any methodknown in the art. For example, multicolor detection and the like arewell known in the art. Instruments for detection of labels are likewisewell known and widely available, e.g., scanners, microscopes, flowcytometers, etc. For example, flow cytometers are widely available,e.g., from Becton-Dickinson ((www.) bd.com) and Beckman Coulter ((www.)beckman.com).

Molecular Biological Techniques

In practicing the present invention, many conventional techniques inmolecular biology, microbiology, and recombinant DNA technology areoptionally used. These techniques are well known and are explained in,for example, Berger and Kimmel, Guide to Molecular Cloning Techniques,Methods in Enzymology volume 152 Academic Press, Inc., San Diego,Calif.; Sambrook et al., Molecular Cloning—A Laboratory Manual (3rdEd.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,2000 and Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (supplemented through2006). Other useful references, e.g. for cell isolation and culture(e.g., for subsequent nucleic acid isolation) include Freshney (1994)Culture of Animal Cells, a Manual of Basic Technique, third edition,Wiley-Liss, New York and the references cited therein; Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (Eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (Eds.)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.

Making Polynucleotides

Methods of making nucleic acids (e.g., by in vitro amplification,purification from cells, or chemical synthesis), methods formanipulating nucleic acids (e.g., by restriction enzyme digestion,ligation, etc.) and various vectors, cell lines and the like useful inmanipulating and making nucleic acids are described in the abovereferences. In addition, methods of making branched polynucleotides(e.g., amplification multimers) are described in U.S. Pat. Nos.5,635,352, 5,124,246, 5,710,264, and 5,849,481, as well as in otherreferences mentioned above.

In addition, essentially any polynucleotide (including, e.g., labeled orbiotinylated polynucleotides) can be custom or standard ordered from anyof a variety of commercial sources, such as The Midland CertifiedReagent Company ((www.) mcrc.com), The Great American Gene Company((www.) genco.com), ExpressGen Inc. ((www.) expressgen.com), Qiagen(oligos.qiagen.com) and many others.

A label, biotin, or other moiety can optionally be introduced to apolynucleotide, either during or after synthesis. For example, a biotinphosphoramidite can be incorporated during chemical synthesis of apolynucleotide. Alternatively, any nucleic acid can be biotinylatedusing techniques known in the art; suitable reagents are commerciallyavailable, e.g., from Pierce Biotechnology ((www.) piercenet.com).Similarly, any nucleic acid can be fluorescently labeled, for example,by using commercially available kits such as those from MolecularProbes, Inc. ((www.) molecularprobes.com) or Pierce Biotechnology((www.) piercenet.com) or by incorporating a fluorescently labeledphosphoramidite during chemical synthesis of a polynucleotide.

REFERENCES

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It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

1.-97. (canceled)
 98. A method of detecting two or more nucleic acidtargets within an individual cell, the method comprising: providing asample comprising the cell, which cell comprises or is suspected ofcomprising two or more nucleic acid targets; providing for each nucleicacid target a label probe system comprising two or more identical labelprobe complexes, each of which comprises a nucleic acid component andone or more labels, wherein the labels of the label probe systems aredistinct for each target nucleic acid; providing for each nucleic acidtarget a capture probe system comprising two or more sets of captureprobes, wherein each set of capture probes comprises two or moredifferent capture probes, wherein all capture probes in the captureprobe system comprise a T section and an L section, wherein the Tsection is a nucleic acid sequence complementary to a section on thenucleic acid target and the L section is a nucleic acid sequencecomplementary to a section on the nucleic acid component of the labelprobe complex, and wherein the T sections of the two or more differentcapture probes are complementary to non-overlapping regions of thenucleic acid target, and the L sections of the two or more differentcapture probes are complementary to nonoverlapping regions of thenucleic acid component of the label probe complex; hybridizing, in thecell and in the presence of cellular non-target nucleic acids, thecapture probe system to the two or more nucleic acid targets, whenpresent in the cell, thereby providing hybridization of two or moredifferent capture probe sets to a single copy of the nucleic acid targetmolecules in the presence of cellular non-target nucleic acids;capturing, in the cell and in the presence of cellular non-targetnucleic acids, the label probe system to the capture probe systemhybridized to the nucleic acid target molecules, wherein the capturingoccurs by simultaneously hybridizing the two or more different captureprobes in each capture probe set to a single molecule of the nucleicacid component of the label probe complex that is complementary to the Lsections of the two or more different capture probes, thereby capturingthe two or more label probe complexes to the nucleic acid target in thepresence of cellular non-target nucleic acids; and detecting a signalfrom the two or more label probe complexes captured on the nucleic acidtargets.
 99. The method of claim 98, wherein hybridizing the two or moredifferent capture probes of the sets of capture probes to the singlemolecule of the nucleic acid component of the label probe complex thatis complementary to the two or more different capture probes isperformed at a hybridization temperature that is greater than a meltingtemperature T_(m) of a complex between each individual capture probe andthe nucleic acid component of the label probe complex.
 100. The methodof claim 98, wherein the two or more different capture probes of thesets of capture probes hybridize to adjacent sections on the nucleicacid target.
 101. The method of claim 98, wherein hybridizing the two ormore different capture probes of the sets of capture probes to thesingle molecule of the nucleic acid target is performed at ahybridization temperature that is greater than a melting temperatureT_(m) of a complex between each individual capture probe and the nucleicacid target.
 102. The method of claim 98, further comprising providingone or more blocking probes capable of hybridizing to regions of thenucleic acid targets not occupied by the two or more different captureprobes of the sets of capture probes.
 103. The method of claim 98,wherein each hybridizing or capturing step is accomplished for each ofmultiple nucleic acid targets at the same time.
 104. The method of claim98, wherein the two or more nucleic acid targets are independentlyselected from the group consisting of a DNA, a chromosomal DNA, an RNA,an mRNA, a microRNA, a ribosomal RNA, a nucleic acid endogenous to thecell, and a nucleic acid introduced to or expressed in the cell byinfection of the cell with a pathogen. targets comprise a first nucleicacid target comprising a first region of an mRNA and a second nucleicacid target comprising a second region of the same mRNA.
 105. The methodof claim 104, wherein one of the two or more nucleic acid targets isDNA.
 106. The method of claim 104, wherein one of the two or morenucleic acid targets is chromosomal DNA.
 107. The method of claim 104,wherein one of the two or more nucleic acid targets is RNA.
 108. Themethod of claim 104, wherein one of the two or more nucleic acid targetsis mRNA.
 109. The method of claim 104, wherein one of the two or morenucleic acid targets is a microRNA.
 110. The method of claim 104,wherein one of the two or more nucleic acid targets is ribosomal RNA.111. The method of claim 104, wherein one of the two or more nucleicacid targets is endogenous to the cell.
 112. The method of claim 104,wherein one of the two or more nucleic acid targets is a nucleic acidintroduced to or expressed in the cell by infection of the cell with apathogen.
 113. The method of claim 98, wherein the two or more nucleicacid targets comprise a reference nucleic acid, and wherein the methodcomprises normalizing the signal of one or more of the nucleic acidtargets to the signal of the reference nucleic acid.
 114. The method ofclaim 98, further comprising the step of correlating the intensity ofthe signal of each nucleic acid target with a quantity of thecorresponding nucleic acid target present in the cell.
 115. The methodof claim 98, wherein the sample comprises a tissue section, or whereinthe sample comprising the cell is derived from a bodily fluid, fromblood, from a swab, or from a cell culture.
 116. The method of claim 98,wherein the cell is in suspension during the hybridizing, capturing,and/or detecting steps.
 117. The method of claim 98, wherein the cell isa circulating tumor cell.
 118. The method of claim 98, wherein the labelprobe system comprises: (A) the two or more identical label probecomplexes each comprising the nucleic acid component and the one or morelabels, wherein the nucleic acid component of the label probe complexescomprises one or more label probes capable of hybridizing to the two ormore different capture probes of the sets of capture probes of thecapture probe system; (B) the two or more identical label probecomplexes each comprising the nucleic acid component and the one or morelabels, wherein the nucleic acid component of the label probe complexescomprises one or more label probes and an amplifier capable ofhybridizing to the one or more label probes and to the two or moredifferent capture probes of the sets of capture probes of the captureprobe system; or (C) the two or more identical label probe complexeseach comprising the nucleic acid component and the one or more labels,wherein the nucleic acid component of the label probe complexescomprises one or more label probes, one or more amplifiers capable ofhybridizing to the one or more label probes, and a preamplifier capableof hybridizing to the one or more amplifiers and to the two or moredifferent capture probes of the sets of capture probes of the captureprobe system.
 119. The method of claim 118, wherein the label probesystem comprises the two or more identical label probe complexes eachcomprising the nucleic acid component and the one or more labels,wherein the nucleic acid component of the label probe complexescomprises one or more label probes capable of hybridizing to the two ormore different capture probes of the sets of capture probes of thecapture probe system.
 120. The method of claim 118, wherein the labelprobe system comprises the two or more identical label probe complexeseach comprising the nucleic acid component and the one or more labels,wherein the nucleic acid component of the label probe complexescomprises one or more label probes and an amplifier capable ofhybridizing to the one or more label probes and to the two or moredifferent capture probes of the sets of capture probes of the captureprobe system.
 121. The method of claim 118, wherein the label probesystem comprises the two or more identical label probe complexes eachcomprising the nucleic acid component and the one or more labels,wherein the nucleic acid component of the label probe complexescomprises one or more label probes, one or more amplifiers capable ofhybridizing to the one or more label probes, and a preamplifier capableof hybridizing to the one or more amplifiers and to the two or moredifferent capture probes of the sets of capture probes of the captureprobe system.